Sperm-egg interaction in mammals is initiated by binding of sperm to the zona pellucida, an acellular coat completely surrounding the plasma membrane of unfertilized eggs. Zonae pellucidae of mouse eggs are composed of three different glycoproteins, designated ZPl, ZP2 and ZP3, having apparent molecular weights of 2DD, DW, 120, DDD and 83, DDD, respectively(Bleil and Wassarman, 1978, lg8Da, 198Db). In this investigation, ZPl, ZP2 and ZP3 were purified from zonae pellucidae isolated individually from unfertilized mouse eggs and 2tell embryos. Each of the glycoproteins was then tested for its ability to interfere with the binding of sperm to eggs in vitro. Solubilized zonae pellucidae isolated from unfertilized eggs, but not from 2cell embryos, reduced binding of sperm to as little as loo/of control values. Similarly, ZP3 purified from zonae pellucidae of unfertilized eggs reduced the binding of sperm to eggs in vitro to an extent comparable to that observed with solubilized zonae pellucidae. On the other hand, ZP3 purified from zonae pellucidae of 2-tell embryos had no significant effect on the extent of sperm binding, consistent with the inability of solubilized zonae pellucidae from 2-tell embryos to affect sperm binding. In no case did purified ZPl and ZP2 interfere significantly with the binding of sperm to eggs in vitro. These results suggest that ZP3 possesses the receptor activity responsible for the binding of sperm to zonae pellucidae of unfertilized mouse eggs. Fertilization apparently results in modification of ZP3 such that it can no longer serve as a receptor for sperm.