Studies of the time course of methotrexate-derived antifolate activity (methotrexate activity) in plasma and skin of rats have been carried out to evaluate the hypothesis that the formation of therapeutically active poly-γ-glutamyl conjugates of methotrexate in skin after systemic administration of the drug will result in the prolongation of methotrexate effects in skin. Such a finding may eventually bear on the use of the drug in the treatment of psoriasis. Three groups of 4 rats received a single i.v. dose of methotrexate, 5, 20, or 40 mg/kg respectively. Serial skin and plasma samples were obtained over 48 h from each rat and analyzed for methotrexate activity by a dihydrofolate reductase enzyme inhibition assay. The values of pharmacokinetic parameters describing the disposition of the drug were calculated from plasma data using standard methods. Skin to plasma ratio of methotrexate activity was also calculated each time a skin sample was obtained. Pharmacokinetic parameter values and skin to plasma ratios were not significantly different between doses. Methotrexate activity declined biexponentially in plasma and skin. The terminal half-life in plasma (mean ± SD) was 20.3 ± 9.2 h. At early times, methotrexate activity in skin was less than plasma, but at later times activity in skin was an order of magnitude greater than that in plasma. In a preliminary study to determine whether the persistent activity in skin is associated with the presence of poly-γ-glutamyl conjugates of methotrexate, a rat was dosed with tritiated methotrexate, 0.18 mg/kg i.v., and skin was obtained 24 h later. Skin homogenate was treated with papain and applied to an anion-exchange column for cleanup prior to injection on a reversed-phase high-pressure liquid chromatography system. Methotrexate poly-γ-glutamates were identified by elution with authentic standards and specific hydrolysis with carboxypeptidase G1.