Abstract The interaction of folic acid antagonists with dihydrofolic reductase was investigated with: (a) the supernatant fraction of chicken liver homogenates; (b) a highly purified enzyme preparation from the same source; and (c) an acetone powder extract of an antifolic-resistant subline of mouse leukemia L1210 possessing high dihydrofolic reductase activity. After incubation with tritiated aminopterin and subsequent dialysis, residual radioactivity per unit of original enzyme activity was the same with the crude and the purified chicken liver dihydrofolic reductase, demonstrating that the inhibitor was not bound appreciably to other proteins. Similarly, inhibition of dihydrofolic reductase activity by graded amounts of aminopterin was the same in both the crude and the purified preparations. The amounts of inhibitor bound per unit of enzyme, as calculated from either the dialysis or inhibit ion experiments, were comparable. Titration of enzyme activity with aminopterin (or with amethopterin) showed that 5.7 × 10 −5 and 6.6 × 10 −5 μmole of inhibitor corresponded to the amount of chicken liver and tumor dihydrofolic reductase, respectively, which had an activity of 1 μmole dihydrofolate reduced per hr at pH 7.5. The inhibition of the chicken liver and tumor enzymes was more pronounced at pH 5.2 than at pH 7.5. It was essentially stoichiometric with excess enzyme, and reversible with equivalent or excess amounts of inhibitor. The previously reported stimulation of dihydrofolic reductase activity by K + and urea was confirmed for both the chicken liver and the tumor enzymes.