Although antifolates such as trimethoprim are used in the clinical treatment of Stenotrophomonas maltophilia infection, the dihydrofolate reductase (DHFR) of this microorganism is scarcely known because it has never been isolated. Here, we describe the purification of this enzyme and kinetically characterize its inhibition by methotrexate (MTX). Upon MTX treatment, time-dependent, slow-binding inhibition was observed due to the generation of a long-lived, slowly dissociating enzyme-NADPH-inhibitor complex. Kinetic analysis revealed a one-step inhibition mechanism (K(I) = 28.9 +/- 1.9 pM) with an association rate constant (k(i)) of 3.8 x 10(7) M(-1)s(-1). Possible mechanisms for MTX binding to S. maltophilia DHFR are discussed.