This experiment was carried out to develop a new technique by immunological method. H-Y antiserum was prepared in inbred Wistar female rats by repeated immunization with new-born testis supernatant and spleen cells from males of the same strain. The activity of H-Y antibody in antiserum was tested by cytotoxicity and biological tests. The results obtained in this experiment. are as follows: In the sperm cytotoxicity test it was showed about 70% of the sperm were dead in the 1/2 to 1/8 dilution of H-Y antiserum immunized with spleen cells, new-born testis or the compound of both antigens. As the dilution increased, the death rate of sperm decreased markedly. H-Y antibody absorbed with female-rat spleen cells showed higher death-rate of sperm than that with male-rat spleen cells. The normal female-rat serum showed the sperm death-rate of 14.6 to 27.9% irrespective of the dilution rate. The difference between the two sperm death-rates was significant. The embryos cultured in the medium of complement and H-Y antiserum immunized with male-rat spleen, new-born testis or the compound of both antigens showed the lysis-rates of 48.9, 50.0 and 46.3% respectively. There was no significant difference among each lysis-rate. But the lysis-rate of the embryos cultured in the medium of complement and normal female rat serum was 5.1% and it was markedly different from the above lysis rates(p $lt;0.001).