The penetration of the zona-pellucida by hamster sperm in vitro (assay drops) was inhibited by aliquots of ultrafiltrates obtained from another set of drops (experimental drops) in which capacitated sperm had interacted with the surface of the zona pellucida. This inhibition was inversely related to the concentration of sperm in the assay drop, and it was noted that the inhibitory response declined when the gametes in the assay drop were incubated together beyond the standard 90-min period. Thus, inhibition was not irreversible. A number of criteria suggest that the inhibitory activity resides in at least some of the peptides (S1 factors) released at four different times after contact between the surfaces of the sperm and zona pellucida. Like the S1 factors, the substances responsible for the inhibitory activity of the supernatants, collected at 2 and 50 min after gamete combination, traversed ultrafilters with Mr cutoffs of 5000 and 2000, respectively, and were inactivated by subtilisin. In addition, the quantity of inhibitory material recovered in the ultrafiltrates from experimental drops containing various numbers of eggs was well correlated with the amount of 2- and 50-min S1 factors from similar experimental drops. These results suggest that the S1 factors are peptide modulators of the penetration process and it is speculated that one of their functions may be to prevent or at least to discourage polyspermic penetration of the hamster zona pellucida.