Methionine synthase (MetH) is a modular protein with at least four distinct regions; amino acids 2-353 comprise a region responsible for binding and activation of homocysteine, amino acids 345-649 are thought to be involved in the binding and activation of methyltetrahydrofolate, amino acids 650-896 are responsible for binding of the prosthetic group methylcobalamin, and amino acids 897-1227 are involved in binding adensylmethionine and are required for reductive activation of enzyme in the cob(II)alamin form. Previous studies have shown that mutations of Cys310 or Cys311 to either alanine or serine result in loss of all detectable catalytic activity. These mutant proteins retain the ability to catalyze methyl transfer from methyltetrahydrofolate to exogenous cob(I)alamin, but have lost the ability to transfer methyl groups from exogenous methylcobalamin to homocysteine [Goulding, C. W., Postigo, D., and Matthews, R. G. (1997) Biochemistry 36, 8082-8091]. We now demonstrate that both MetH holoenzyme and a truncated MetH(2-649) protein, which lacks a cobalamin prosthetic group, contain 0.9 equiv of zinc, while the Cys310Ser and Cys311Ser mutant proteins contain less than 0.05 equiv of zinc. Addition of l-homocysteine to MetH(2-649) is accompanied by release of 1 equiv of protons/mol of protein, while addition of l-homocysteine to the Cys310Ser and Cys311Ser mutant truncated proteins does not result in proton release. The Cys310Ala and Cys311Ala mutant methylcobalamin holoenzymes have completely lost the ability to transfer the methyl group from methylcobalamin to homocysteine, suggesting that zinc is required for this reaction. Further evidence that zinc is required for catalytic activity comes from experiments in which the zinc is removed from MetH(2-1227). Removal of zinc from methylated wild-type holoenzyme by treatment with methyl methanethiolsulfonate and then with dithiothreitol and EDTA results in loss of the ability of the protein to catalyze methyl transfer from methyltetrahydrofolate to homocysteine. Reconstitution of the zinc-depleted holoenzyme results in incorporation of 0.4 equiv of zinc/mol of protein and partial restoration of the ability of the protein to catalyze homocysteine methylation.