In this investigation, the interaction of mouse sperm with unfertilized eggs and embryos, solubilized zonae pellucidae isolated from eggs and embryos, and purified zona pellucida glycoproteins ZP1, 2, and 3 (J. D. Bleil, and P. M. Wassarman, (1980b)Dev. Biol.76, 185–202) has been examined in vitro by light and electron microscopy. The experiments described were carried out in order to determine the temporal sequence of events during sperm-egg interaction in vitro and to identify the component(s) of zonae pellucidae responsible for inducing mouse sperm to undergo the acrosome reaction. “Pulse-chase” analysis of the sequence of sperm-egg interactions revealed that mouse sperm first “attach” loosely and then “bind” tightly to the unfertilized egg's zona pellucida. Binding of sperm to egg zonae pellucidae is followed by induction of the acrosome reaction. Induction of the acrosome reaction can be mediated by the zona pellucida, since solubilized zonae pellucidae isolated from unfertilized eggs were found to be just as effective as the calcium ionophore A23187 in inducing the reaction in vitro. Furthermore, ZP3 purified from zonae pellucidae isolated from unfertilized eggs, but not from two-cell embryos, was also just as effective as either solubilized zonae pellucidae from eggs or ionophore A23187 in inducing the acrosome reaction. ZP1 and 2 from both eggs and embryos, and ZP3 from embryos, had little effect on the extent of the acrosome reaction as compared to control samples. The results of these and other experiments (J. D. Bleil, and P. M. Wassarman, (1980b)Cell20, 873–882) strongly suggest that, at least in vitro, mouse sperm recognize and bind to ZP3 of egg zonae pellucidae, and that such binding leads to the induction of the acrosome reaction. Modification of ZP3 following fertilization eliminates sperm binding to zonae pellucidae and, consequently, induction of the acrosome reaction is precluded.