The metabolism of methotrexate in rabbits was investigated following 6 hr of infusion with [3',5',7-(3)H]methotrexate (50 mg/kg). Methotrexate and its metabolites were analyzed by an enzyme kinetic method, reverse phase high-performance liquid chromatography, and scintillation counting of radioactivity. 7-Hydroxymethotrexate was found to be a major metabolite in plasma, urine, bile and various tissues of rabbits. Cumulative production of 7-hydroxymethotrexate during the first 6 hr was 31.8% of the total dose. The peak 7-hydroxymethotrexate concentration in plasma was reached at the end of the infusion, and this concentration was higher than the methotrexate concentration. The plasma clearance of 7-hydroxymethotrexate was biphasic in nature and slower than that of methotrexate. The highest methotrexate concentration was found in kidney after 6 hr. This concentration was found to be 7.2 times greater than that in plasma. A higher ratio of 7-hydroxymethotrexate/methotrexate was found in liver, small intestine, kidney and testis when compared to plasma. Lung and kidney showed significant conversion of methotrexate to 7-hydroxymethotrexate in vitro, as did the liver.