Several chemotherapeutic protocols for the treatment of malignancies include administration of methotrexate (MTX) during or shortly after total anesthesia. Clinical observations in patients treated for breast carcinoma or childhood cancer have shown unexpected myelosuppression and mucosal damage. This phenomenon may be attributed to the synergistic effects of nitrous oxide, which inactivates the cobalamin coenzyme of methionine synthase, and MTX, which inhibits dihydrofolate reductase, on folate metabolism. However, no quantitative data on dose-effect relationships are available regarding the combined toxicity of MTX and N2O. We investigated the effect of exposure to N2O on the toxicity of MTX. Groups of male Wistar rats were exposed to either 50% N2O/50% O2 or air for 12-48 h. Subsequently, a single i.p. injection of 10, 20, 40, or 80 mg MTX/kg body weight was given. Gastrointestinal toxicity resulted in diarrhea and weight loss in all groups for 5 days after MTX administration. Concomitantly, bone marrow depression with leukocytopenia and thrombocytopenia occurred. Exposure to N2O did not alter the plasma clearance of MTX. No substantial liver or kidney toxicity could be detected, but the 50% lethal dose for MTX was reduced from 60 mg/kg to 10 mg/kg if rats had been exposed to N2O for 48 h; the main causes of death were dehydration and bleeding. The administration of 5-formyl-tetrahydrofolate (4 x 10 mg i.p.) but not 5-methyltetrahydrofolate protected completely against the lethal effect of the drug combination. Altogether, cytotoxic effects of MTX on proliferating cells are potentiated by N2O. Therefore, the use of this anesthetic shortly before or during MTX administration should be avoided.