In mammals, methionine synthase plays a central role in the detoxification of the rogue metabolite homocysteine. It catalyzes a transmethylation reaction in which a methyl group is transferred from methyltetrahydrofolate to homocysteine to generate tetrahydrofolate and methionine. The vitamin B 12 cofactor cobalamin plays a direct role in this reaction by alternately accepting and donating the methyl group that is in transit from one substrate (methyltetrahydrofolate) to another (homocysteine). The reactivity of the cofactor intermediate cob(I)alamin renders the enzyme susceptible to oxidative damage. The oxidized enzyme may be returned to the catalytic turnover cycle via a reductive methylation reaction that requires S-adenosylmethionine as a methyl group donor, and a source of electrons. In this study, we have characterized an NADPH-dependent pathway for the reductive activation of porcine methionine synthase. Two proteins are required for the transfer of electrons from NADPH, one of which is microsomal and the other cytoplasmic. The cytoplasmic protein has been purified to homogeneity and is soluble cytochrome b 5 . It supports methionine synthase activity in the presence of NADPH and the microsomal component in a saturable manner. In addition, purified microsomal cytochrome P450 reductase and soluble cytochrome b 5 reconstitute the activity of the porcine methionine synthase. Identification of soluble cytochrome b 5 as a member of the reductive activation system for methionine synthase describes a function for this protein in nonerythrocyte cells. In erythrocytes, soluble cytochrome b 5 functions in methemoglobin reduction. In addition, it identifies an additional locus in which genetic polymorphisms may play a role in the etiology of hyperhomocysteinemia, which is correlated with cardiovascular diseases.Homocysteine is a rogue metabolite that is generated by the hydrolysis of S-adenosylhomocysteine, the spent form of the ubiquitous methyl group donor S-adenosylmethionine. Elevated levels of plasma homocysteine constitute a significant and independent risk factor for cardiovascular diseases (1-7). There are two major metabolic avenues for detoxifying homocysteine in mammalian cells. Transmethylation, catalyzed by methionine synthase or by betaine-homocysteine methyltransferase, salvages homocysteine as methionine, whereas transsulfuration, catalyzed by cystathionine -synthase, commits it to degradation (Fig. 1). Of these three enzymes, betaine-homocysteine methyltransferase has a very limited tissue distribution and has been found only in the liver and kidney (8). Mutations in either methionine synthase (9, 10) or cystathionine -synthase (11) result in severe hyperhomocystinemia, with attendant early and aggressive occlusive arterial diseases. Noticing the correlation between prominent arterial damage and elevated homocysteine, McCully (12) boldly postulated in 1969 that the vascular alterations could be attributed to high homocysteine concentrations.Despite the longevity of this hypothesis, little is know...