The glycosidase-recognizing N-acetylglucosamine terminal residue, N-acetylglucosaminidase (NAG), has been repetitively implicated in fertilization. Nevertheless, its role in the multiple steps comprising this process is a matter of debate because it has been involved in zona pellucida (ZP) binding and penetration and polyspermy block. In this study, the involvement of NAG during sperm interaction with the ZP was analysed. Soluble ZP was able to inhibit sperm NAG activity, suggesting that it can be recognized as a ligand by this enzyme. Sperm-ZP binding assays were carried out under conditions where acrosome reaction (AR) could not take place (salt-stored oocytes and a modified medium where Ca(2+) was replaced by Sr(2+)). Different NAG-specific reagents-an inhibitor (2-acetamido-2-deoxy-D-glucono-1,5-lactone), a substrate (p-nitrophenyl-N-acetylglucosaminide) and an anti-NAG antibody-were able to impair sperm binding to the ZP when present during these assays. The lactone was also able to inhibit oocyte penetration during IVF assays, although not when present after primary binding had taken place. This result was not related to the interference of lactone with AR or zona penetrability. Exogenous NAG also inhibited sperm-oocyte interaction when present during binding and IVF assays or used for oocyte pre-incubation. These results suggest the participation of NAG in sperm primary binding to the ZP.