Methionine dependency has been reported in cancer cell lines and primary tumors. Thus, L-methionine deprivation might have potential value for the treatment of human cancers with a methionine requirement. L-Methionine-alpha-deamino-gamma-mercaptomethane-lyase has been reported to decrease plasma methionine levels and to inhibit tumor growth in experimental animals but has not been studied extensively because sufficient homogeneous enzyme was not available. In this study, we cloned the L-methioninase gene from Pseudomonas putida and isolated pure and abundant recombinant enzyme. Both L-methionine and L-cysteine in culture medium were completely degraded by 1 unit/ml purified enzyme. Two hundred and fifty units/kg L-methioninase administered i.v. to mice yielded 0.7 unit/ml of plasma concentration and lowered total plasma sulfur-containing amino acids by more than 75%. Although sensitivity to enzymatic methionine depletion differed among cell lines, leukemia cell lines were generally more sensitive than solid tumor cell lines. The availability of pure recombinant L-methioninase will allow in vivo studies on the antitumor activity and the potential toxicity of enzymatic methionine depletion.