The zona pellucida is an extracellular matrix surrounding the mammalian egg where species-specific gamete recognition and signaling occur. Pig zona pellucida were isolated in large amounts and used as an affinity matrix for detergent-solubilized, biotinylated membrane proteins of pig spermatozoa. On non-reducing SDS-polyacrylamide gel electrophoresis, specifically bound sperm proteins migrated with M(r) 170,000, 150,000, 130,000, 56,000, and 50,000 (p50). Disulfide bond reduction separated each of the M(r) 130,000-170,000 proteins into M(r) 105,000 (p105) and M(r) 45,000 (p45) subunits, indicating that these high M(r) proteins are related. Based on two-dimensional electrophoresis, the M(r) 56,000 band was composed of three to four proteins that migrated with M(r) 56,000-62,000 (p56-62) in the second (reducing) dimension. p50 bound to heterologous zona pellucida (murine, bovine) and to Xenopus laevis oocyte envelopes, demonstrating a lack of species specificity to its binding and was identified as proacrosin/acrosin based on amino acid sequences of two tryptic peptides and its interaction with monospecific antibodies to proacrosin. In contrast, p105/p45 and one or more of the p56-62 proteins bound to pig zona pellucida but not to the egg extracellular matrices of the other species; these proteins therefore exhibited the species-specific binding to the zona pellucida expected for molecules involved in specific gamete adhesion. Amino acid sequences of nine tryptic peptides derived from p105/p45 did not match peptide sequences in existing databases, establishing it as a unique protein. These (p105/p45 and at least one p56-62 protein) are the first sperm membrane proteins to be identified that bind in a species-specific manner to the egg extracellular matrix.