Characteristics of methotrexate (MTX) inhibition of dihydrofolic acid reductase (DHFR) enzyme activity and the effects of NADPH and NADH on enzyme-drug interaction were studied. Two highly sensitive assay procedures were used. The first utilized tritium-labeled MTX to measure direct binding properties of the enzyme and the second utilized tritium-labeled dihydrofolate (H2PteGlu) and folate (PteGlu) to analyze kinetics of reduction of these substrates. NADPH was found to enhance DHFR binding of MTX (Kd = 2.6 X 10(-11) M), whereas NADH was found to have no effect (Kd = 3.7 X 10(-9) M). However, NADH proved to be a good substrate for folate reduction compared to NADPH, especially in low salt buffer. The observation that NADH supports the reduction of folate and dihydrofolate but not MTX binding suggests that natural resistance to MTX could exist if NADH replaces NADPH as the main cofactor for DHFR.