Methotrexate was found to stimulate asparagine synthetase activity in vivo by approximately six-fold in rat liver. The maximum effect of methotrexate on hepatic asparagine synthetase activity was observed sixteen hours after intraperitoneal injection of the drug. Cycloheximide, like methotrexate, is a protein synthesis inhibitor and was used to determine that asparagine synthetase activity was not preferentially stimulated under stress. As expected, hepatic asparagine synthetase activity falls markedly with the decreased protein synthesis caused by injection of cycloheximide. It is proposed that methotrexate inhibits serine-dependent glycine biosyn-thesis by decreasing the concentration of tetrahydrofolate for serine hydroxymethyltransferase. This leads to a stimulation of asparagine synthetase to provide nitrogen for asparagine-dependent glycine synthesis. This may provide an explanation of the observed chemotherapeutic synergism between asparaginase and methotrexate treatment.