The specificity of early sperm-zona interactions in vitro was studied using gametes from the mouse, rabbit, pig, and human. Mouse epididymal spermatozoa, capacitated in vitro, bound to the zonae of intact mouse oocytes and zona fragments removed mechanically from oocytes. When viewed with the scanning electron microscope (SEM), spermatozoa were found adherent more frequently to the external (cumulus) surface of the zonae than the internal (oocyte) surface. Noncapacitated mouse spermatozoa or spermatozoa incubated with zonae in the absence of external calcium did not bind to homologous zonae. by contrast, fresh, epididymal mouse spermatozoa and mouse spermatozoa capacitated in vitro appeared to attach equally well to both surfaces of zonae and zona fragments from rabbit and pig oocytes. Rabbit spermatozoa "capacitated" in vivo as well as noncapacitated rabbit spermatozoa, attached rapidly to both inner and outer surfaces of zonae from rabbit, mouse, and pig oocytes. This reaction was not dependent on external calcium. Human spermatozoa did not display any tendency to adhere to zonae from the rabbit, mouse, or pig. The results of these studies suggest that spermatozoa can associate with zona in a nonspecific as well as a specific manner. The use of heterologous systems in addition to homologous systems in sperm binding assays may be useful in distinguishing these two types of interaction.