Mouse intra-acrosomal antigens, recognized by monoclonal antibodies, MN7 and MC41, were analyzed by SDS-PAGE and Western blotting. Both the MN7- and MC41-antigens were glycoproteins of 90kDa and 200kDa, respectively. However, the two antigens exhibited different biochemical properties; the MN7-antigen was extracted only in the acidic condition (pH 3.0), while the MC41-antigen was detectable in both acidic and neutral conditions. The releasing process during the acrosome reaction was examined by immunoelectron microscopy. The MN7-antigen, which was initially distributed uniformly in the entire acrosome, gradually disappeared from the acrosome, being localized once at the cortex-medulla border region at the middle stage, and dispersed without close contact with the vesiculated membrane. By contrast, the MC41-antigen, which was initially restricted to the cortical region of the anterior acrosome, was localized on the electrondense materials lining closely to the fenestrated membrane at the middle stage, and dispersed keeping in close contact with the vesicles. These findings indicate that the antigens originally segregated into different subdomains can be differentially released during the acrosome reaction.