Surface antigens of mammalian sperm were studied by use of monoclonal antibodies (MAs). Six hybridoma cell lines were obtained by fusion of mouse myeloma cells with spleen cells from rats immunized with unwashed, epididymal sperm from C3H mice. Quantitative assessment of antibody binding, using a solid phase, antibody-protein A assay, indicated that four MAs bound to integral, sperm surface antigens; two others bound to nonintegral sperm antigens or epididymal fluid components. Immunofluorescence studies showed specific binding of individual MAs to localized regions: acrosome, midpiece, and midpiece and tail. All of these MAs inhibited sperm-egg binding, and those to the midpiece and/or tail immobilized sperm cells. The monoclonal antibodies provide probes for immunochemical characterization of sperm antigens and for elucidation of the role of the antigens in sperm.