Isoantibodies to whole semen or epididymal spermatozoa of rabbits inhibit fertilization in vitro. This inhibition is attributed to a primary blocking of 1 or more sperm antigens essential for the sperm-egg interaction and not to a secondary action (e.g., agglutination, complement-dependent immobilization), because univalent (Fab [antibody active fragments]) antibody fragments, which did not agglutinate or immobilize spermatozoa, were also effective. Hyaluronidase, the sperm enzyme required for penetration of the cumulus surrounding the egg, could be one such sperm antigen. This possibility was supported by the fact that cumulus dispersion was inhibited in previous experiments and that hyaluronidase-inhibiting antibodies were present in isoantisera to rabbit semen. It is necessary to employ antibody preparations specific for rabbit sperm hyaluronidase to examine critically the inhibition of fertilization by antihyaluronidase antibodies.