Many of nitric oxide's biological effects are mediated via NO binding to the iron in heme-containing proteins. Cobalamin (vitamin B 12 ) is structurally similar to heme and is a cofactor for methionine synthase, a key enzyme in folate metabolism. NO inhibits methionine synthase activity in vitro, but data concerning NO binding to cobalamin are controversial. We now show spectroscopically that NO reacts with all three valency states of cobalamin and that NO's inhibition of methionine synthase activity most likely involves its reaction with monovalent cobalamin. By following incorporation of the methyl moiety of [ 14 C]methyltetrahydrofolic acid into protein, we show that NO inhibits methionine synthase activity in vivo, in cultured mammalian cells. The inhibition of methionine synthase activity disrupted carbon flow through the folate pathway as measured by decreased incorporation of [ 14 C]formate into methionine, serine, and purine nucleotides. Homocysteine, but not cysteine, attenuated NO's inhibition of purine synthesis, providing further evidence that NO was acting through methionine synthase inhibition. NO's effect was observed both when NO donors were added to cells and when NO was produced physiologically in co-culture experiments. Treating cells with an NO synthase inhibitor increased formate incorporation into methionine, serine, and purines and methyl-tetrahydrofolate incorporation into protein. Thus, physiological concentrations of NO appear to regulate carbon flow through the folate pathway.Vitamin B 12 deficiency leads to pernicious anemia and subacute combined degeneration of the spinal cord (1). Pernicious anemia is characterized by megaloblastic erythropoiesis and is secondary to decreased activity of methionine synthase, one of two mammalian enzymes that requires vitamin B 12 (cobalamin) as a cofactor (2, 3). Methionine synthase catalyzes the transfer of the methyl group of 5-methyltetrahydrofolate to homocysteine via a methylcobalamin intermediate with cycling of cobalamin between the ϩ1 valency state (i.e. cbl(I)) 1 and the ϩ3 valency state (i.e. cbl(III)) (2, 3). Methyltetrahydrofolate is the major intracellular storage form of folates, and its synthesis from 5,10-methylene tetrahydrofolate is essentially irreversible in vivo (2, 4) (Fig. 1). Thus, decreased methionine synthase activity leads to trapping of intracellular folates as 5-methyltetrahydrofolate, and the megaloblastic anemia of vitamin B 12 deficiency is virtually indistinguishable from the megaloblastosis of folate deficiency (1).Nitric oxide (NO) is produced by most cell types and regulates a diverse array of biological functions (5, 6). NO has been reported to inhibit methionine synthase activity in vitro (7-9), and it might be expected to bind to the cobalt in cobalamin because (i) NO binds tightly to the iron in heme (10); (ii) ferrous heme and cbl(III) are isoelectronic; and (iii) in both heme and cobalamin, the metal ion is coordinated to four in-plane nitrogen atoms of a tetrapyrrole ring and has two out-of-plane ligands (3...