Abstract: To investigate the pharmacokinetics of the active metabolite of methotrexate (MTX), 7‐hydroxy‐methotrexate (7‐OH‐MTX), 1 mg/kg of the compound was administered as single intravenous bolus injections to 6 unanaestetized rats. For comparison, 1 mg/kg of the parent drug MTX was given to the same number of animals. Venous blood samples were drawn at intervals for a total of 120 min., and thereafter the rats were sacrificed and tissue samples were obtained from the brain, lungs, liver, kidneys, testes, fat, and muscle. Pharmacokinetics of 7‐OH‐MTX and MTX were biphasic, with a significantly (P < 0.05) smaller central compartment of distribution (Vc) and a longer second phase half‐life (t1/2(β)) for 7‐OH‐MTX. MTX tissue concentrations exceeded those of 7‐OH‐MTX in all tissues examined. The highest 7‐OH‐MTX concentrations were found in renal tissue. It is implied that 7‐OH‐MTX is less extensively distributed than the parent compound.