By using microassay, the strong human sperm agglutination were caused by 9 of 23 monoclonal antibodies (McAbs) obtained by immunization with fresh human acrosome-reacted (AR) sperm, which was induced by ionophore A 23187, but no one of them demonstrated the ability to immobilize human sperm. In contrast to most studies reported by other laboratories, the antigens recognized by most of McAbs in the present study were located in equatorial segment and midpiece of human sperm, and no one in acrosome region of human sperm. The binding pattern of some McAbs on non-treated and AR sperm was found to be different. The human sperm proteins ranged in the size of 16-146 kDa were demonstrated to be reacted with 9 of 23 McAbs by immunoblotting. Moreover, the cross reaction of 23 McAbs with mouse and tree shrew sperm was detected by indirect immunofluorescent assay. These results suggest that AR sperm possesses peculiar immunogenic properties.