H-Y antigen has been defined by various immunological methods including graft rejection, T-cell mediated cytolysis, and antiserum cytotoxicity. The H-Y phenotype is normally associated with the male sex in mammals. Using specific T-cell clones, two Y-linked genes coding for H-Y epitopes have been identified in the mouse, Smcy and Uty, and one of these genes, SMCY, is also present on the human Y chromosome. Anti-H-Y antisera detect a membrane bound antigen associated with beta2-microglobulin, and a soluble protein secreted by testicular Sertoli cells. The membrane bound antigen appears to be different from the peptides detected by cytotoxic T-cells, and for the soluble antigen evidence was provided that, in mammals it may be identical with anti-Müllerian hormone. In non-mammalian vertebrates, serological H-Y antigen is associated with the heterogametic sex, and sex-reversal of the homogametic sex results in the occurrence of H-Y antigen. Originally, H-Y antigen was believed to be responsible for sex determination, thus representing the testis-determining factor (TDF) in mammals, but this hypothesis has been disproved by showing that male gonadal differentiation can also occur (in the mouse) in the absence of H-Y antigen. In the meantime, SRY is considered to be TDF. Although it was revealed that the H-Y antigens as detected by different assays are genetically heterogeneous, they are probably involved in male-specific functions in mammals, and possibly in the differentiation of the heterogametic sex in non-mammalian vertebrates.