The inhibitory action of cholesterol-containing suspensions on fertilizing capacity in uterine-capacitated rabbit sperm cells showed a direct dependence on the concentration of sterol. Dispersion with synthetic phosphatidylcholine as a nonsonicated suspension or as liposomes did not alter this antifertilization effect. Esterification of the sterol, however, caused a complete loss of inhibitory activity. Cholesterol inhibited induction of the acrosome reaction among epididymal rat spermatozoa incubated under chemically defined conditions. Other agents with a negative effect on the acrosome reaction were seminal plasma membrane vesicles and palmitic acid. Egg lecithin-liposomes and bovine serum albumin, especially after being delipidated, facilitated it. These results corroborate the viewpoint that changes in the lipid bilayer of sperm plasma membrane significantly influence fertilizing capacity among mammalian spermatozoa.