Sperm penetration of zonae and fertilization of mouse eggs in vitro was studied as a function of sperm concentration over the range 10(3)-10(6) cells/ml. Maximal fertilization was obtained at 10(5) sperm/ml with markedly reduced levels seen at lower concentrations. Eggs inseminated at low sperm concentrations were, however, characterized by the absence of multiple sperm penetrations of their zonae and by the retention of an intact cumulus oophorus during culture. Penetration levels were similar in eggs inseminated with and without an intact cumulus, although increases in supernumerary and supplemental sperm were observed with partly denuded ova. The distribution of sperm found within eggs inseminated in vitro was compared with a distribution predicted by chance alone. The similarity in observed and expected distributions indicated that a functional zona reaction was absent in eggs inseminated in vitro. Sperm binding to zonae of cumulus-free unfertilized mouse eggs was shown to be related to sperm concentration over the range 10(3)-10(6) cells/ml. Zonae sperm binding sites, while too numerous for accurate quantitation, exceeded 100 per zona. The interrelationships between sperm penetration of and sperm binding to the zonae of cumulus-free eggs were examined. At low sperm concentrations, the ratio of sperm/zonae with bound sperm to sperm/penetrated egg approached one, although the total number of bound sperm exceeded the total number of penetrating sperm. That proportion of cells in a preincubated epididymal sperm suspension, capable of binding to the zona was determined and employed in an estimation of the extent of sperm capacitation.