Pig zona pellucida (ZP) contains three families of glycoproteins: PZP2, PZP3 alpha and PZP3 beta. PZP3 alpha mediates the binding of the ZP to spermatozoa. In this study, the binding site of pig ZP on boar spermatozoa and the zona-binding proteins of boar spermatozoa were studied using chemically modified zona glycoproteins or anti-pig ZP antiserum. Endo-beta-galactosidase-digested PZP3 alpha (E beta G-PZP3 alpha), which is deficient in sulfated N-acetylpolyactosamine, as well as solubilized ZP, bound to the acrosomal region of acrosome-damaged or partially acrosome-reacted spermatozoa. However, they did not bind to acrosome-intact or fully acrosome-reacted spermatozoa. Solubilized ZP did bind to the acrosomal cap released upon acrosome reaction. In western blot analyses, E beta G-PZP3 alpha bound to the sperm proteins with molecular masses similar to proacrosin-acrosin and the binding was inhibited by fucoidan and anti-pig acrosin antiserum. These results suggest that the binding site of solubilized pig ZP and E beta G-PZP3 alpha on spermatozoa is located mainly in the acrosomal matrix and on the membranous compartments in the acrosome and suggest that E beta G-PZP3 alpha binds to proacrosin-acrosin. The binding of E beta G-PZP3 alpha to proacrosin-acrosin may be involved in the binding of the ZP to the acrosome of partially acrosome-reacted spermatozoa.