These experiments were carried out to clarify the optimalconditions and interspecific cross reaction of H-Y antibody activity. H-Y antiserum was prepared in inbred SD female rats and Balb/c female mice by repeated immunization of rat newborn testis homogemate, rat and mouse spleen cells obtained from males of same strain. The activity of H-Y antibody in antiserum was tested by ELISA and biological tests. The cross reactivity of H-Y antibody was confirmed by culturing mouse and rabbit embryos in medium containing H-Y antibody and complement obtained from rat and guinea pig, respectively. The optimal condition for the activity of H-Y antibody was also investigated by culturing embryos in medium with different pH and complement concentration. The results obtained in these experiments were summarized as follows: 1. The formation rates of H-Y antibody in rats immunized with newborn testis and spleen cell were 40.0 and 50.0% respectively, and that in mouse immunized with spleen cell was 48.4%. 2. The activity of H-Y antibody was not affected by pH in range of 6.5 to 8.0, and the same was true for the relative concentration of complement to the H-Y antibody. 3. Minimum time needed for the activity of H-Y antibody was confirmed to be 0.5 to 1 hour and 24 to 48 hours respectively for the zona free embryos and intact embryos. 4. When mouse and rabbit embryos were treated with H-Y antibody obtained from rat, 46.4 and 54.8% of embryos were retarded or destroyed. From these results it could be said that H-Y antibody had strong interspecific cross reactivity.