Abstract : The chemical reduction of methemoglobin (metHb) with dithionite and other reducing agents has proven unsatisfactory due to undesirable byproducts. We have now developed a method to reduce methemoglobin on a preparative scale, in which metHb is reduced using photoactivated flavin mononucleotide (FMN). The reduction is performed in the absence of oxygen, since oxygen is reduced more rapidly than metHb and leads to the formation of H2O2. DL-methionine was routinely used as the electron donor, although a variety of compounds could act in this capacity. Thus, a 1% metHb solution containing 20 Mm DL-methionine and 20 micro mol FMN was reduced by more than 95% in 30 min using a 300-watt light bulb. The reaction rate depended on the methionine concentration as well as on the light intensity. The reaction was independent of pH between pH 6 and 8.