The kinetic characteristics of a purified insect dihydrofolate reductase (DHFR) have been described. TheKmvalues for the substrate dihydrofolate and the cofactor NADPH have been estimated by primary and secondary Hanes plots to be 0.3 and 5.2 μM, respectively.Drosophila melanogasterDHFR can use folate and NADH at acidic pH values, but at a much lower rate than the preferred substrate and cofactor. Folic acid is a partial competitive inhibitor ofDrosophilaDHFR (Ki = 0.4 μM) and trimethoprim is a complete competitive inhibitor (Ki = 5.4 μM). Methotrexate binds less tightly to theDrosophilaenzyme than to many other DHFRs (Kd = 0.9 nM).DrosophilaDHFR is inhibited by KCl and organic mercurials and is slightly activated by urea. These data indicate thatDrosophilaDHFR has some characteristics which are typical of vertebrate DHFRs and others which are typical of prokaryotic DHFRs. The study of this enzyme, therefore, should aid in the definition of the structural features that are responsible for the kinetic characteristics in different DHFRs.Key words: dihydrofolate reductase,Drosophila melanogaster, methotrexate.