Kinetic studies were performed to evaluate the interaction of benzamidine (BD), 4-aminobenzamidine (ABD), 4''-nitrophenyl 4-guanidinobenzoate (NPGB) and 4''-methylumbelliferyl 4-guanidinobenzoate (MUGB) with mouse acrosin. The Michaelis constant of mouse acrosin towards .alpha.-N-benzoyl-L-arginine ethyl ester and the sensitivity of mouse acrosin to inhibitors differed from those reported for other species. NPGB and MUGB were much more active inhibitors of acrosin than BD and ABD. Plots of percentage fertilization vs. acrosin inhibitor concentration were generated for all 4 compounds. Linear dose-response curves were obtained and gave ED50 values (50% inhibition of fertilization) of 230 .mu.M for BD, 27 .mu.M for ABD, 35 nM for MUGB and 13 nM for NPGB. The relative antifertility activity of the compounds paralleled their inhibitory activity towards mouse acrosin, strongly indicating that the inhibiton of fertilization is obtained through the inhibition of acrosin. Since the dose-response curves were linear, the mouse in-vitro fertilization system may be useful to screen acrosin inhibitors for their infertility potency. MUGB should have low toxicity and may have potential as a contraceptive agent.