The avian perivitelline membrane (PVM) is the site of initial contact between sperm and egg. It consists of only two major components, which are both homologues of the mammalian zona pellucida (ZP) proteins, and belong to the ZP1 and ZPC families, respectively. We have established a method to isolate large quantities of both native avian ZP proteins and have used these preparations to investigate their sperm-binding capacities. Chicken ZPC forms multimeric structures of defined size and binds to an approximately 180-kDa protein complex present in rooster sperm extracts. Based on experiments using both PVM and isolated proteins, we show that chicken ZP1 is proteolytically degraded by a sperm-associated protease but that chicken ZPC remains intact. An antiserum directed against chicken ZP1 is capable of inhibiting sperm binding to the PVM. Taken together, these data suggest that ZP1, in addition to ZPC, plays a major role in the initial interactions between sperm and egg.