Seminal plasma prevents human sperm from becoming acrosomally responsive. These experiments tested the idea that the inhibitory activity of seminal plasma is contained in the particulate prostasome fraction. Most of the inhibitory activity was sedimentable (105,000 g, 2 h) and the majority of the recovered activity was in the prostasome fraction. The recovery of inhibitory activity in the prostasome fraction (42% of the activity in unfractionated seminal plasma) was similar to the recovery of cholesterol in that fraction (41%), consistent with cholesterol's role as the major inhibitor in seminal plasma. To test whether components of the prostasome fraction bind to sperm, the prostasome fraction was made fluorescent with fluorescein isothiocyanate or with the acetoxymethyl ester of 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. No labeled material was seen to bind to sperm, suggesting that exchange of cholesterol between prostasomes and sperm takes place through the aqueous phase or at the time of vesicle-sperm collision.