ZPB, one of the pig zona pellucida glycoproteins, can be purified after removal of sialylated and/or sulfated N-acetylpolylactosamine from the nonreducing region of its carbohydrate chains by digestion with endo-beta-galactosidase. Among the components produced, only ZPB shows sperm-binding activity after the digestion. Recently, we have shown that N-linked carbohydrate chains of endo-beta-galactosidase-digested ZPB (EbetaG-ZPB) are predominantly involved in sperm binding [Yonezawa, N., Aoki, H., Hatanaka, Y. & Nakano, M. (1995) Eur. J. Biochem. 233, 35-41]. In this study, to define the sperm-binding region in EbetaG-ZPB, glycopeptides were purified from lysyl endopeptidase digests of EbetaG-ZPB and analyzed for sperm-binding activity by an in vitro competition assay. The locations of the glycopeptides were determined from partial amino acid sequences, amino acid and sugar composition analyses, and apparent molecular masses after SDS/PAGE. The N-terminal fragment (amino acid residues 137-247), which contains two N-linked carbohydrate chains, showed a significant inhibition of sperm-egg binding. However, the fragment that had one N-linked carbohydrate chain (residues 325-341) and the fragment that had two or three O-linked carbohydrate chains (residues 248-324) did not inhibit sperm-egg binding. Thus, the two N-linked carbohydrate chains in the N-terminal fragment of EbetaG-ZPB are important for sperm binding of pig zona pellucida.