We have examined the metabolism of the folate analog methotrexate (MTX) in the human parasite Leishmania major. These cells readily hydrolyzed MTX to N-10-methyl-4-deoxy-4-aminopteroate (MAPA), such that following a 24-h incubation in 1 microM [3H]MTX approximately 30% of the cell-associated radioactivity was MAPA. MAPA also accumulated in the culture medium, exceeding the concentration of MTX after 24 h. Neither 7-hydroxy-methotrexate nor MTX polyglutamates were observed in cells or medium, even after a 72-h incubation with MTX. In contrast to MTX, folate is extensively polyglutamylated in L. major (Santi, D. V., Nolan, P., and Shane, B. (1987) Biochem. Biophys. Res. Commun. 146, 1089-1092). MAPA was found to be 190-fold less potent than MTX as an inhibitor of leishmanial growth and to bind less tightly than MTX to leishmanial dihydrofolate reductase-thymidylate synthase. We therefore examined the possibility that MTX resistance is mediated by increased MTX hydrolysis to MAPA in drug-resistant Leishmania. However, enzymatic assays show that the rate of MTX hydrolysis was unaltered in the MTX-resistant R1000-3 line and the primaquine-resistant PQ-R30 line (which is 24-fold cross-resistant to MTX). In addition to MAPA, several minor unidentified metabolites were observed in the LT252, R1000-5B, and PQR30 cells but no consistent differences in the amounts of these metabolites were evident among these lines. These data indicate that alterations in the rate of MTX hydrolysis in vitro, or in the characteristics of MTX metabolites formed in vivo, do not underly the MTX resistance displayed by the H region-amplified R1000-5B and PQ-R30 lines.