Monoclonal antihuman sperm antibodies were used to study the surface antigens of sperm from man, monkey (Macaca mulatta), dog, rabbit, bull and mouse. Quantitative absorption results indicate some of the antibodies cross-react with sperm of all the species tested while three recognized determinants were unique to the human sperm. These reactions were sperm-specific since no cross-reactivity was observed with somatic tissue from human and mouse. In the indirect immunofluorescence test, monoclonal antibodies MA1 and MA4 recognize antigenic determinants which appear within the mouse testis during spermatogenesis and are present on the acrosome and mid-piece sperm regions, respectively, of the species tested. MA2 reacted diffusely with the acrosome of human and monkey sperm only. In contrast, MA3 had a speckled acrosomal staining pattern reacting only with human sperm. Antibodies MA5 and MA6 were also specific for human sperm, binding to the equatorial and tail regions. Live human and monkey sperm were agglutinated and immobilized by MA1 but only immobilized by MA2 . With the Western blot technique, human sperm antigens of approximately 84,000, 240,000, 30,000 and 71,000 daltons were identified by antibodies MA1 , MA3 , MA4 and MA5 , respectively. An acrosomal antigen of approximately 82,000 daltons present on both bull and mouse sperm was identified by MA1 . Antibody MA4 , in turn, identified at 28,000-dalton mouse sperm antigen and a 35,000- to 37,000-dalton antigen present on the midpiece of bull sperm.