Fertilizing capacity among uterine-capacitated rabbit sperm cells declined exponentially during incubation with membrane vesicles from seminal plasma. In suspensions containing an average of 0.42 mg vesicle protein/10(6) sperm, decapacitation occurred with a half-time of 23 min (ki (native vesicles) = 1.78 +/- 0.14 h-1). Exposing these membrane vesicles to pronase retarded decapacitation, prolonging its half-time to 51 min (ki (pronase-digested vesicles) = 0.81 +/- 0.06 h-1). Cholesterol-bearing liposomes suppressed sperm-fertilizing capacity at a comparable rate. In suspensions containing an average of 0.52 mg lipid/10(6) sperm, decapacitation had a half-time also of 51 min (ki (liposomes) = 0.82 +/- 0.14 h-1). These lower inhibition rates accompanied diminished rates of vesicle uptake by spermatozoa. Membrane vesicles labeled with phosphatidyl[14C]choline rapidly bound to epididymal sperm cells, displaying a half-time of 2.3 min (ka (native vesicles) = 18.0 +/- 0.35 h-1). Following pronase treatment, this interval increased to 17 min (ka (pronase-digested vesicles) = 2.48 +/- 0.37 h-1). Liposome binding data yielded a half-time of 28 min (ka (liposomes) = 1.47 +/- 0.17 h-1). Postbinding decapacitation half-times for these vesicles, given by the difference between binding and decapacitation intervals, appear broadly alike: native vesicles, 21 min, pronase-digested vesicles, 34 min, and liposomes, 23 min. During this interval, a vesicle antifusigen (cholesterol) apparently transfers to the sperm plasma membrane inhibiting the acrosome reaction. The lipid bilayer in these membrane vesicles withstood proteolytic attack, as seen by electron microscopy. Pronase acted principally to hydrolyze vesicle glycoproteins, which evidently bind to the sperm surface during decapacitation.