Monoclonal antibodies (mAbs) to the rabbit sperm autoantigens RSA-1, 2 and 3 were found to cross-react with human ejaculated spermatozoa. The mAbs show by indirect immunofluorescent staining that the antigen is concentrated in the middle-piece region of the tail, with lesser amounts on the head region. Binding of mAbs to human sperm extract was 75% of the maximum binding to rabbit spermatozoa. Immunoadsorbent chromatography and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) identified three sperm antigens of 87,000, 15,000 and 13,000 daltons (kd). The mAbs significantly inhibited human spermatozoa from penetrating zona-free hamster oocytes. It is concluded that human sperm, like rabbit sperm, may have a low molecular weight family of membrane glycoproteins with a higher molecular weight extrinsic protein bound together in a complex on their surfaces.