Monoclonal antibodies were raised against acrosomal antigens of human sperm by immunizing BALB/CA mice with purified ejaculated human spermatozoa. An ELISA-assay, employing glutaraldehyde-fixed spermatozoa as antigen, was used to screen the hybridomas producing anti-human sperm antibodies. Two hybridoma cell-lines produced antibodies which bound to the acrosomal region of spermatozoa. Both gave identical results in preliminary tests and therefore only one was chosen for further experiments. This antibody stained the acrosomal region of fixed but not living spermatozoa by indirect immunofluorescence, indicating an intra-acrosomal localization of the antigen. In acetone-fixed frozen sections of human testis this antigen was expressed only in germ cells in the adluminal compartment of seminiferous tubules. The antigen was clearly visible in round spermatids from the beginning of the cap phase of acrosome development and was also present in premature germ cells which were present in ejaculates and which were in the early stages of acrosome development. By immunochemical analysis this antibody recognized a molecule of 50 K MW as well as other components of 24 to 34 K. The pattern of staining for the antigen was similar in the presence or absence of beta-mercaptoethanol in the sample buffer. The species specificity of the antigen was studied by indirect immunofluorescence using acetone-fixed spermatozoa and the antigen was found to be present in mouse, bovine, ram and boar spermatozoa. This antibody may be useful as an acrosomal marker.