The Science of Methylene Blue: Coal Tar, Redox Chemistry and the Antidote Years

Methylene blue is one of the oldest synthetic substances still used in hospitals. Heinrich Caro made it in 1876 as a dye for cotton, and fifteen years later Paul Guttmann and Paul Ehrlich gave it to two malaria patients in Berlin. Unlike most of the early medicines on this site, it did not come from a plant, a mould or a mineral spring. It came out of coal tar, the black residue of the gas works, by way of the chemistry that turned coal-tar compounds into colours. What makes it a medicine is a piece of chemistry that also makes it a dye: the molecule flips easily between a blue form and a colourless form, giving and taking electrons as it goes.

This page is about that chemistry and the medical uses that grew from it. It explains where the dye comes from and how it differs from the natural dyes and plant medicines of its time, how the blue and colourless forms work, what the dye does inside red blood cells, why it turns brown blood red again in methaemoglobinaemia, and how a Berkeley physiologist, Matilda Moldenhauer Brooks, proposed it in 1932 as an antidote to cyanide and carbon monoxide. It follows the debate that proposal set off in the 1930s, a 2018 laboratory re-examination of the cyanide effect, a 24-year record from a New York poison centre, and the blue urine that has marked the drug since its earliest medical use. The lives of Caro and Guttmann, the 1891 malaria report and the modern brain and mitochondria research each have their own page in this wing.

Table of Contents

  1. From Coal Tar to a Thiazine Dye
  2. Natural Dyes and Natural Medicines in the Background
  3. Blue and Colourless: The Redox Couple
  4. How the Dye Behaves in Red Blood Cells
  5. Methaemoglobinaemia and Its Reversal
  6. Matilda Moldenhauer Brooks and the 1932 Cyanide and Carbon Monoxide Studies
  7. The 1930s Antidote Debate
  8. A Modern Re-examination of the Cyanide Effect
  9. Twenty-Four Years of Poison-Centre Experience
  10. Blue Urine and Other Visible Signs
  11. Key Research Papers
  12. Connections
  13. Featured Videos

1. From Coal Tar to a Thiazine Dye

In the nineteenth century, cities lit their streets with gas made by heating coal. The process left behind coal tar, a thick, smelly mixture that was at first a nuisance. Chemists learned to distil it into simpler compounds, among them benzene. Benzene could be turned into nitrobenzene and then into aniline, and aniline and its relatives became the raw material of a new industry. In 1856 William Henry Perkin made the first commercial aniline dye, mauve. Heinrich Caro spent his Manchester years, from 1859 to 1866, improving its production before he returned to Germany in 1866 and, from 1868, led research at the dye works in Ludwigshafen.

Methylene blue belongs to this family of coal-tar colours. Its starting material is dimethylaniline, an aniline carrying two small methyl groups on its nitrogen. Caro’s route, in outline, built a larger molecule from two such units and closed a ring containing a sulphur atom between them. The result was a bright blue dye that took well to cotton, a fibre that many early aniline dyes coloured poorly. Caro made it in 1876, and in 1877 a German patent was granted for it, described by the company as Germany’s first patent for a coal-tar dye.

What kind of molecule it is

Chemically, methylene blue is a thiazine dye: its core is three rings fused in a row, with a sulphur atom and a nitrogen atom in the middle ring. The same three-ring core, without the charged dye groups, is called phenothiazine. In its usual form, methylene blue is a salt with a chloride counter-ion, and its formal name in pharmacology is methylthioninium chloride. It dissolves in water to give an intense blue solution; a very small amount colours a large volume of water. The exact arrangement of its atoms was worked out not by Caro but by the chemist August Bernthsen, whose long “Studies in the Methylene Blue Group” appeared in 1885. Bernthsen later wrote Caro’s memorial biography.

The molecule’s simplicity matters for everything that follows. Reviewers of its medical history, the chemists Mark Wainwright and Kate Crossley, put the range of its uses down to “the combination of its simple chemical structure and facility for oxidation-reduction reactions” inside the body. The phenothiazine core went on to become the starting point for a whole line of later medicines, a story told on the From Dye to Drug page.

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2. Natural Dyes and Natural Medicines in the Background

Methylene blue has no natural source. No plant, mould or animal makes it; every gram ever used was made in a factory. That sets it apart from most of the medicines in this Pharmacology section, which began as willow bark, foxglove leaf, opium latex or cinchona bark. But the natural world is very much in the background of its story, in two ways.

The natural dyes the industry set out to replace

Before coal tar, cloth was coloured with plants and animals. The red of madder came from the root of the madder plant, whose main colouring substance is alizarin. The deep blue of indigo came from the leaves of indigo plants and woad. In 1868 the German chemists Carl Graebe and Carl Liebermann made alizarin in the laboratory, the first natural dye to be made synthetically; the historian Anthony Travis describes that synthesis as a milestone both for chemical theory and for the growth of the synthetic dye industry. Caro worked with Graebe and Liebermann to turn their laboratory method into a factory process, patented in 1869–70, and synthetic alizarin went on to displace the madder crop. Indigo took far longer: Adolf von Baeyer made it in the laboratory between 1878 and 1880, Caro directed the long industrial effort at Ludwigshafen, and a factory process succeeded only in 1897, after Caro had left the research post.

Methylene blue was not a copy of any natural dye. It was a new colour with no counterpart in nature, the kind of molecule the coal-tar chemists could make once they understood how dyes were built.

The plant medicine it was measured against

The medical background was a plant too. For more than two centuries before 1891, malaria in Europe was treated with the bark of the South American cinchona tree, and from 1820, when the French pharmacists Pierre-Joseph Pelletier and Joseph-Bienaimé Caventou isolated quinine from the bark, with quinine itself. When Guttmann and Ehrlich gave methylene blue to their two malaria patients in 1891, the comparison was with this plant alkaloid. The systematic review by Lu and colleagues (2018) calls methylene blue “the first synthetic antimalarial to be discovered”, used “against all types of malaria” in the late nineteenth and early twentieth centuries. Quinine, the plant medicine, nonetheless remained the main treatment of the period, and the history of cinchona is told on the site’s History of Malaria page.

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3. Blue and Colourless: The Redox Couple

The most useful fact about methylene blue is that it exists in two forms that turn into each other easily. The familiar blue form is the oxidised form. When it accepts electrons (chemically, two electrons and a hydrogen ion), it becomes leucomethylene blue, from the Greek leukos, “white”, because this reduced form is colourless. When leucomethylene blue gives those electrons away again, for example to oxygen, it turns back into the blue form.

A pair of substances like this, one able to take electrons and the other able to give them, is called a redox couple (from reduction and oxidation). Chemists have long used methylene blue as an indicator for exactly this reason: a solution that loses its blue colour is a sign that something in it is giving electrons away, and the colour coming back is a sign that oxygen or another oxidising agent has arrived. A classic school demonstration, sometimes called the “blue bottle” experiment, shows the colour disappearing in a sugar solution and returning when the bottle is shaken with air.

Why this makes it a shuttle

Because the dye can be reduced and re-oxidised over and over, a small amount can carry many electrons from one place to another. It acts less like a fuel that is used up than like a shuttle bus. Inside the body, cells supply electrons to the blue form, and the colourless form then hands those electrons on to whatever will accept them. Which partner receives them depends on where the dye is and how much of it is present. In red blood cells, as the next sections explain, the most important partner is the iron in haemoglobin. In mitochondria, the energy-producing compartments of cells, the dye can pass electrons into the chain of proteins that uses oxygen — the basis of the modern research described on the Methylene Blue Today page.

The same property can work in either direction. In large amounts the blue form can itself pull electrons away from other molecules, acting as an oxidising agent. Pushparajah Mak and Liebelt, in a 2021 review of methylene blue as an antidote, note that its action rests partly on its oxidising ability — “ironically the same mechanism” that causes the blood disorder it is used to treat. That double edge runs through the rest of this page.

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4. How the Dye Behaves in Red Blood Cells

Red blood cells are bags of haemoglobin, the protein that carries oxygen. Each haemoglobin unit holds an iron atom at its centre. To carry oxygen, that iron has to be in its ferrous state (Fe2+). If it loses one more electron and becomes ferric iron (Fe3+), the protein becomes methaemoglobin, which cannot carry oxygen and which makes the remaining normal haemoglobin hold on to its oxygen more tightly, so less is released to the tissues.

A small amount of methaemoglobin forms every day in healthy people, and red cells have their own repair system. The main pathway uses an enzyme that draws electrons from a carrier molecule called NADH and passes them back to the iron. A second, normally minor pathway uses a different carrier, NADPH, and an enzyme often called NADPH-methaemoglobin reductase. On its own this second enzyme does very little, because it has no natural partner to hand the electrons to.

The dye supplies the missing partner

Methylene blue fills that gap. Inside the red cell, the NADPH-dependent enzyme reduces the blue dye to colourless leucomethylene blue. Leucomethylene blue then gives its electrons to the ferric iron of methaemoglobin, turning it back into ferrous iron and the protein back into working haemoglobin. The dye returns to its blue form, ready to be reduced again. In effect, the dye switches on a repair pathway that is normally idle.

Where the NADPH comes from, and why G6PD matters

Red cells make their NADPH through a short chain of reactions whose first step is carried out by the enzyme glucose-6-phosphate dehydrogenase, or G6PD. People with inherited G6PD deficiency, one of the most common enzyme disorders in the world, make less NADPH in their red cells. For them, the dye has less fuel to work with, and because NADPH is also the cell’s main defence against oxidising stress, the oxidising side of the dye can damage the cells instead, breaking them open (haemolysis). The New York poison-centre series described in section 9 recorded that G6PD activity was deficient in two of the seven patients tested, and the pooled analysis of four malaria trials in West African children by Müller and colleagues (2013) found a small fall in haemoglobin in children with full G6PD deficiency and two episodes of haemolysis among 1,005 children. Those authors judged the finding of limited clinical relevance while noting a need for monitoring. The site’s G6PD Deficiency page covers the condition itself.

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5. Methaemoglobinaemia and Its Reversal

When more than a small share of the blood’s haemoglobin is in the methaemoglobin form, the condition is called methaemoglobinaemia (American spelling: methemoglobinemia). Most cases are acquired: something swallowed, inhaled or given as a medicine oxidises haemoglobin faster than the red cells can repair it. Blood with a lot of methaemoglobin looks chocolate-brown rather than red, and the skin and lips take on a blue-grey colour that does not improve with extra oxygen. As the level rises, the lack of oxygen delivery causes headache, breathlessness, confusion and, at high levels, collapse.

The New York series in section 9 shows which substances were most often involved in one large city: volatile nitrites (41 per cent of cases), local anaesthetics (15 per cent) and the antibiotic dapsone (11 per cent). Certain other medicines and some chemicals in industry and in contaminated well water are also recognised causes.

The dye as the standard antidote

Methylene blue is the treatment most associated with this condition. Pushparajah Mak and Liebelt describe it as “best known as an antidotal treatment for acquired methemoglobinemia”. Given into a vein, it works through the NADPH pathway described above, and in most reported cases the brown colour of the blood and the blue-grey of the skin fade within an hour or so as the iron is restored. The same review describes newer uses that extend beyond methaemoglobinaemia, in a nerve-toxic reaction to the cancer drug ifosfamide and in a form of shock in which blood vessels fail to respond to the usual drugs.

When the treatment can backfire

The redox chemistry sets limits. In people with G6PD deficiency the dye may work poorly or cause haemolysis, for the reasons in section 4. And because the blue form is itself an oxidising agent, very large amounts can push the balance the other way and contribute to methaemoglobin formation. Separately, methylene blue is a potent inhibitor of the enzyme monoamine oxidase A; Ramsay, Dunford and Gillman (2007) showed this in the laboratory, explaining reports of serotonin toxicity in patients who received the dye while taking certain antidepressants. Those safety findings are covered in more detail on the site’s Methylene Blue: Methemoglobinemia and G6PD Deficiency page and on this wing’s Methylene Blue Today page.

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6. Matilda Moldenhauer Brooks and the 1932 Cyanide and Carbon Monoxide Studies

For forty years after Guttmann and Ehrlich’s malaria report, methylene blue was known in medicine mainly as a stain and as an antimalarial that newer synthetic drugs were gradually replacing. Its move into emergency medicine began with a woman physiologist at the University of California, Berkeley: Matilda Moldenhauer Brooks, whose research concerned oxidation–reduction dyes and living cells.

The 1932 animal report

In June 1932 Brooks published a short report, “Effect of Methylene Blue on CN and CO Poisoning”, in the Proceedings of the Society for Experimental Biology and Medicine (the journal now called Experimental Biology and Medicine). CN is the chemical shorthand for cyanide and CO for carbon monoxide. Both poisons stop the body using oxygen, but in different ways. Cyanide blocks cytochrome oxidase, the last protein in the mitochondrial chain that hands electrons to oxygen, so cells cannot use the oxygen even when it reaches them. Carbon monoxide binds tightly to haemoglobin in place of oxygen, so less oxygen reaches the cells in the first place. Brooks reported that methylene blue counteracted both kinds of poisoning in her animal experiments.

The 1933 JAMA letter

In January 1933 she brought the idea to physicians in a short item in the Journal of the American Medical Association, “Methylene blue as antidote for cyanide and carbon monoxide poisoning”. The proposal reached doctors at a moment when cyanide poisoning, from fumigants, industrial processes and suicide, had no well-established antidote. The dye was tried in poisoned patients in the early 1930s, but the details of the first clinical uses are not documented in the sources this page relies on, so they are not repeated here.

Why the dye was expected to work

The explanation usually given at the time, and repeated in later textbooks, ran through methaemoglobin. Cyanide binds strongly to ferric iron, including the ferric iron of methaemoglobin. If an antidote turned some of the blood’s haemoglobin into methaemoglobin, that methaemoglobin could act as a sponge, pulling cyanide out of the cells to form a harmless-for-the-moment compound called cyanmethaemoglobin. On this reasoning, the oxidising side of methylene blue was the useful one. As section 8 explains, a 2018 study concluded that this was not, in fact, how the dye protected its animals.

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7. The 1930s Antidote Debate

Brooks’s proposal set off a quick and public argument. Within months, other researchers and physicians were writing to journals about whether the dye worked, how well, and whether better antidotes existed. The surviving record is a run of short papers and letters, several of them without abstracts, so what follows reports who wrote and what their titles addressed rather than claims about details that cannot be checked.

How the argument ended

The combination of a nitrite with sodium thiosulphate, the approach studied by Chen’s group, became the standard cyanide antidote for most of the twentieth century: the nitrite forms methaemoglobin to draw cyanide out of the cells, and thiosulphate helps the body convert cyanide into the much less toxic thiocyanate, which is passed in the urine. Methylene blue dropped out of cyanide treatment. The 2018 Haouzi study describes research on its mechanism and efficacy against cyanide as having been “abandoned for decades”. For carbon monoxide, oxygen therapy remained the main treatment; the site’s Carbon Monoxide Poisoning page covers that condition.

Methylene blue did not leave emergency medicine, however. Its role simply shifted to the condition its chemistry suits best: reversing methaemoglobinaemia, including methaemoglobinaemia caused by too much nitrite.

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8. A Modern Re-examination of the Cyanide Effect

In 2018 a team led by Philippe Haouzi at Pennsylvania State University College of Medicine, with collaborators at Temple University and in Paris, went back to Brooks’s observation. They were prompted, they wrote, by recent studies of methylene blue in injuries that follow a lack of oxygen.

What they did

The work was done in rats and in a line of human cells grown in the laboratory. The team first tested how the rats’ circulation and metabolism responded to a range of doses, finding the dye harmless up to 50 mg per kilogram of body weight in their animals. They then infused nine rats with a lethal dose of potassium cyanide and treated them with either salt water or methylene blue at 20 mg/kg, a dose they estimated corresponds to roughly 4 mg/kg in humans. A further ten rats received the dye five minutes after a smaller, non-lethal cyanide exposure. In the cells, they measured energy (the ATP/ADP ratio), harmful reactive oxygen molecules, the electrical charge across the mitochondrial membrane, and oxygen use, before and after adding the dye to cyanide-poisoned cells.

What they found

The cyanide infusion killed every untreated rat within seven to eight minutes. In the rats given methylene blue at the same time, blood pressure and heart contraction were restored, the oxygen deficit was limited, and all the animals survived — without any significant methaemoglobinaemia. Given after a non-lethal exposure, the dye sped the recovery of blood lactate and oxygen balance. In the cells, it reduced reactive oxygen production and restored the energy ratio, membrane charge and oxygen use.

What it changed

The absence of methaemoglobin was the key finding. The authors concluded that the antidote effect “cannot be accounted for by the creation of a cyanomethemoglobinemia” — the explanation given in the 1930s — and that it seemed instead to come from the dye’s redox properties, which, depending on the dose, could directly counter some of cyanide’s effects on cell metabolism. In plain terms, the dye seemed to help the poisoned cells’ energy machinery keep running, which fits its role as an electron shuttle in mitochondria. The study was in animals and cells; it is a laboratory finding, not evidence from treated patients, and the authors framed it as a reason to consider the question again.

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9. Twenty-Four Years of Poison-Centre Experience

Methylene blue has been the accepted treatment for acquired methaemoglobinaemia for decades, yet the authors of the largest recent case series noted that “data regarding clinical outcomes are sparse”. In 2025 Rothenberg, Biary and Hoffman, medical toxicologists at the NYU Grossman School of Medicine and the New York City Poison Center, published every case reported to that centre between 2000 and 2024 in which the dye was given for methaemoglobinaemia.

What the series contained

What happened to the patients

Improvement after the dye was reported in 98 per cent of cases. Adverse effects judged to be caused by the dye were reported in nine cases (4.9 per cent), including one case of haemolysis. G6PD activity was tested in only seven patients; it was deficient in two, and one of those two did not improve. Two patients died, both after exposure to sodium nitrite. The authors concluded that in their series the dye was “both efficacious and well tolerated”, with a single dose of 1–2 mg/kg enough for most patients, and that major adverse effects were extremely rare — while noting how few patients had been tested for G6PD deficiency.

The doses above are reported as the findings of a hospital case series, given by clinicians to patients with a measured blood disorder. They are a record of what one poison centre observed, more than a century after Caro first made the dye for cotton.

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10. Blue Urine and Other Visible Signs

Methylene blue announces itself. Much of a dose leaves the body through the kidneys, partly as the colourless leucomethylene blue, which turns back to the blue form on contact with air. The urine is coloured blue or blue-green. The 2018 systematic review of malaria treatment by Lu and colleagues lists “blue coloration of urine” among the effects recorded across 21 studies and 1,504 patients, alongside mild urinary and digestive symptoms.

Other signs

Because the dye is so intensely coloured, it can tint other things too: the stool, the saliva and, at higher doses given into a vein, the skin, which may look bluish. That bluish tint can be confused with the blue-grey of low oxygen. The colour also absorbs red light strongly, and clinical reviews note that it can make finger-clip pulse oximeters, which read oxygen levels by shining red and infrared light through the finger, report falsely low values for a while after a dose. These colour effects are a direct consequence of the chemistry in section 3: the oxidised dye absorbs red-orange light and so looks deep blue.

A colour with a history

The visible blue has been part of the drug’s story from the beginning. Ehrlich valued methylene blue because its colour showed where it went in living tissue: it stained nerve fibres in 1886 and the malaria parasite soon after. More than a century later, the colour is still the simplest evidence that the dye Heinrich Caro made for cotton has been given as a medicine.

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Key Research Papers

  1. Wainwright M, Crossley KB. Methylene Blue--a therapeutic dye for all seasons? J Chemother. 2002;14(5):431-43. PubMed PMID: 12462423
  2. Bernthsen A. Studien in der Methylenblaugruppe. Justus Liebigs Annalen der Chemie. 1885;230(1):73-136. DOI: 10.1002/jlac.18852300106
  3. Travis AS. Chemical Modeling: From Paul Ehrlich’s Dyes to β-Blockers-A Brief History. J Comput Biol. 2019;26(7):726-734. PubMed PMID: 31066580
  4. Lu G, Nagbanshi M, Goldau N, Mendes Jorge M, Meissner P, Jahn A, Mockenhaupt FP, Müller O. Efficacy and safety of methylene blue in the treatment of malaria: a systematic review. BMC Med. 2018;16(1):59. PubMed PMID: 29690878
  5. Pushparajah Mak RS, Liebelt EL. Methylene Blue: An Antidote for Methemoglobinemia and Beyond. Pediatr Emerg Care. 2021;37(9):474-477. PubMed PMID: 34463662
  6. Müller O, Mockenhaupt FP, Marks B, Meissner P, Coulibaly B, Kuhnert R, Buchner H, Schirmer RH, Walter-Sack I, Sié A, Mansmann U. Haemolysis risk in methylene blue treatment of G6PD-sufficient and G6PD-deficient West-African children with uncomplicated falciparum malaria: a synopsis of four RCTs. Pharmacoepidemiol Drug Saf. 2013;22(4):376-85. PubMed PMID: 23135803
  7. Ramsay RR, Dunford C, Gillman PK. Methylene blue and serotonin toxicity: inhibition of monoamine oxidase A (MAO A) confirms a theoretical prediction. Br J Pharmacol. 2007;152(6):946-51. PubMed PMID: 17721552
  8. Brooks MM. Effect of Methylene Blue on CN and CO Poisoning. Experimental Biology and Medicine (then Proc Soc Exp Biol Med). 1932;29(9):1228-1229. DOI: 10.3181/00379727-29-6295
  9. Brooks MM. Methylene blue as antidote for cyanide and carbon monoxide poisoning. JAMA. 1933;100(1):59. DOI: 10.1001/jama.1933.02740010061028
  10. Hanzlik PJ. Subject of This Letter: Methylene Blue as Antidote for Cyanide Poisoning. Cal West Med. 1933;38(3):225-6. PubMed PMID: 18742459
  11. Draize JH. Sodium tetrathionate and methylene blue in cyanide and carbon monoxide poisoning. Science. 1933;78(2016):145. PubMed PMID: 17778489
  12. Chen KK, Rose CL, Clowes GHA. Methylene Blue, Nitrites, and Sodium Thiosulphate against Cyanide Poisoning. Experimental Biology and Medicine (then Proc Soc Exp Biol Med). 1933;31(2):250-251. DOI: 10.3181/00379727-31-7079p
  13. Geiger JC. Concerning cyanide antidotes. Cal West Med. 1935;43(6):457. PubMed PMID: 18743484
  14. Haouzi P, Gueguinou M, Sonobe T, Judenherc-Haouzi A, Tubbs N, Trebak M, Cheung J, Bouillaud F. Revisiting the physiological effects of methylene blue as a treatment of cyanide intoxication. Clin Toxicol (Phila). 2018;56(9):828-840. PubMed PMID: 29451035
  15. Rothenberg R, Biary R, Hoffman RS. Effectiveness and tolerability of methylthioninium chloride (methylene blue) for the treatment of methemoglobinemia: twenty-four years of experience at a single poison center. Clin Toxicol (Phila). 2025;63(4):284-291. PubMed PMID: 40062661

PubMed Topic Searches

  1. Methylene blue and methemoglobinemia
  2. Methylene blue and cyanide poisoning
  3. Leucomethylene blue (the colourless reduced form)
  4. Methylene blue and G6PD deficiency
  5. History of methylene blue

Further Reading

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