SummaryWe have previously reported that rats fed on a vitamin B 12 (B 12 )-deficient diet containing 180 g soybean protein per kg diet showed marked histologic damage in their testes. In this paper, we report the effect of B 12 -deficiency on B 12 -dependent methionine synthase in the rats' testes and the effect of methionine supplementation of the diet on testicular damage. Rats were fed the soybean protein-based B 12 -deficient diet for 120 d. We confirmed that those rats were in serious B 12 -deficiency by measuring urinary methylmalonic acid excretion and B 12 content in tissues. Methionine synthase activity in the testis of the B 12 -deficient rats was less than 2% of that in B 12 -supplemented (control) rats. To complement disrupted methionine biosynthesis, methionine was supplied in the diet. A supplement of 5 g D,L-methionine per kg diet to the B 12 -deficient diet did not affect urinary methylmalonic acid excretion of B 12 -deficient rats. The testicular histology of rats fed the methioninesupplemented B 12 -deficient diet was almost indistinguishable from that of control rats. Thus, we conclude that the lowered testicular methionine synthase activity is the primary cause of the histologic damage due to B 12 -deficiency and that methionine supplementation to the diet can reduce the damage. These findings would indicate the importance of the methionine synthase activity, especially for testicular function. Key Words vitamin B 12 -deficiency, rat, testis, methionine synthase In mammals, vitamin B 12 (B 12 ) acts as a cofactor for two enzymes, methylmalonyl-CoA mutase and methionine synthase. Adenosylcobalamin is required for methylmalonyl-CoA mutase, which catalyzes a reaction that interconverts succinyl-CoA and methylmalonylCoA. Dysfunction of methylmalonyl-CoA mutase causes methylmalonic aciduria. Methylcobalamin (MeB 12 ) binding to methionine synthase serves as an intermediary in methyl transfer from methyltetrahydrofolate to homocysteine, donating the methyl group to homocysteine to form methionine, and then accepting a methyl group from methyltetrahydrofolate to form tetrahydrofolate. Methionine synthase is important both to resynthesize methionine and to metabolize methyltetrahydrofolate. Tetrahydrofolate accepts C1-groups from serine or formate. Then, the C1-group can be donated to either homocysteine to form methionine or deoxyuridine monophosphate to form deoxythymidine monophosphate. The C1-group also is precursor in de novo purine biosynthesis. Methionine is one of the amino acids that are building blocks for protein. In addition, methionine is activated to S -adenosylmethionine (AdoMet), which is well known as a major methyldonor for numerous substrates and an allosteric regulator for methylenetetrahydrofolate reductase (MTHFR) and cystathionine- -synthase. While causal of symptoms related with dysfunction of methionine synthase, such as megaloblastic anemia and neuropathy, might be explained by the "Methyl trap" hypothesis ( 1-4 ), the biochemical mechanism has not yet been fully understoo...