Some murine melanomas and hepatocellular carcinomas (HCCs) have been shown to be auxotrophic for arginine. Arginine deiminase (ADI; EC 3.5.3.6.), an arginine-degrading enzyme isolated from Mycoplasma, can inhibit growth of these tumors. We found that ADI was specific for arginine and did not degrade other amino acids. Although arginine is not an essential amino acid for most cells, all human melanomas and HCCs tested were found to be inhibited by ADI in vitro. Arginine is synthesized from citrulline in two steps by argininosuccinate synthetase and argininosuccinate lyase. Melanomas and HCCs did not express argininosuccinate synthetase mRNA but did express argininosuccinate lyase mRNA, suggesting that the arginine auxotrophy of these cells was a result of an inability to produce argininosuccinate synthetase. Human melanomas and HCCs were transfected with an expression plasmid containing argininosuccinate synthetase cDNA. The transfected cells were much more resistant to ADI than the parental cells in vitro and in vivo. Initial attempts to use ADI in vivo indicated that this enzyme had little efficacy, consistent with its short circulation half-life. Formulation of ADI with polyethylene glycol to produce ADI-SS PEG_{20,000 mw} resulted in an enzyme with a much longer circulation half-life that, and although equally effective in vitro, was more efficacious in the treatment of mice implanted with human melanomas and HCCs. These data indicate that sensitivity of melanoma and HCC is due to the absence of argininosuccinate synthetase in these cells and that an effective formulation of ADI, which causes a sustained decrease in arginine, may be a useful treatment for arginine auxotrophic tumors including melanoma and HCC.