The elevated dependence on methionine of tumor cells is a cancer-specific metabolic defect. In current studies, the recombinant L-methionine alpha,gamma-lyase (rMETase), an L-methionine depleting enzyme cloned from Pseudomonas putida, was shown to have efficacy in a broad series of cancer cell lines. Twenty-one different human tumor cell lines (4 lung, 4 colon, 4 kidney, 4 melanoma, 3 CNS, and 2 prostate) from the NCI Human Tumor Cell Line Screen and 7 human normal cell strains were treated with rMETase in vitro. We showed that rMETase had mean IC(50) (units rMETase/ml) for the following cancer cell types: renal cancer, 0.07; colon cancer, 0.04; lung cancer, 0.12; prostate cancer, 0.01; melanoma, 0.19; and CNS cancer, 0.195, which was approximately one order of magnitude lower than that for normal cell strains: skin fibroblasts, 4; aortic smooth muscle cells, 0.88; aortic endothelial cells, 0.8; keratinocytes, 0.75, and bronchial epithelial cells, 0.75. rMETase was also conjugated with polyethylene lycol (PEG). PEG-rMETase also had high cell-kill activity. In vitro studies, animal studies and clinical trials have now shown that methionine restriction is an effective anticancer strategy. Cells from many different types of cancers are methionine dependent. The most effective strategy to deplete methionine is with the use of rMETase. PEG-rMETase offeres additional advantages of increasing the circulating half-life and reducing the immunogenicity of rMETase which is a bacterial protein. The results of the current study demonstrate the broad clinical potential for rMETase and rMETase for cancer treatment.