Acrosin, an enzyme required for fertilization, is an endogenous proteinase associated with membranes of the sperm acrosome. Liposomes were utilized as a model system to evaluate the mode of association between highly purified boar acrosin and phospholipid bilayer membranes. Acrosin was observed to bind to liposomes containing acidic phospholipids such as phosphatidylglycerol, cardiolipin, and phosphatidylserine. There was no apparent binding of acrosin to liposomes which consisted of nonacidic phospholipids, thus indicating that an ionic phospholipid constituent was required for binding. Increased ionic strength caused a significant reduction in acrosin-liposome association with an inverse effect on enzyme-membrane dissociation. Acrosin-liposome association and dissociation were similarly effected by increasing concentrations of divalent cations at constant ionic strength, and by reductions in pH. Equilibrium binding experiments, with anionic liposomes, suggest the presence of either multiple classes of independent binding sites, or apparent negative cooperativity, with a range in the apparent affinity constant (Ka) from 2 x 10(11) M-1 at low acrosin concentrations to 3 x 10(8) M-1 at high acrosin concentrations. The membrane-associated enzyme was accessible to concanavalin A, and to high molecular weight substrates, demonstrating that a portion of the acrosin molecule is exposed at the membrane surface. In addition, acrosin binding to liposomes had no apparent effect of hydrolysis of soluble protein or synthetic substrates. The results demonstrate that acrosin-liposome binding is due in part to electrostatic charge interactions and indicate that the enzyme has properties of an extrinsic membrane protein.