Experimental conditions were established for an investigation of the sperm binding characteristics of the mouse zona pellucida. Fresh epididymal sperm binding to zonae of unfertilized eggs was a time-dependent process, reflecting the occurrence of sperm capacitation under in vitro incubation conditions. In contrast, capacitated sperm binding in large numbers to zonae of unfertilized ova occurred independently of temporal considerations. A dramatic difference was apparent in the binding of capacitated sperm to zonae of unfertilized eggs (>100 per zona) and to in vivo produced 2-cell embryos (0-5 per zona), providing a basis for assessing the occurrence of a zona reaction. Under comparable conditions, capacitated sperm bound in large numbers to zonae of in vitro produced 2-cell embryos. Possible reasons for a subnormal or inadequate zona reaction in eggs inseminated in vitro have been discussed.