Elevated dependence on methionine of cancer cells is a cancer-specific metabolic defect. Recombinant L-methionine alpha,gamma-lyase (rMETase), an L-methionine depleting enzyme cloned from Pseudomonas putida, was shown to have efficacy on 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 a mean IC(50) (units rMETase/ml) for the following cancer cell types: renal, 0.07; colon, 0.04; lung, 0.12; prostate, 0.01; melanoma, 0.19; and CNS, 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 glycol (PEG). PEG-rMETase also had high cancer cell-killing 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 cancer are methionine dependent. The most effective strategy to deplete methionine is with the use of rMETase. PEG-rMETase offers 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 PEG-rMETase for cancer treatment.