HLA-B7 by 50%(IC50) was 34 nM, where-as the IC50 for the SMCX peptide was 140 nM (Fig. 3B). Thus, the significant differ-ence in the ability of the SMCY and SMCX peptides to sensitize targets for T cell rec-ognition is almost entirely due to the fine specificity of the T cell receptor, rather than the differences in MHC binding affin-ities. The SMCX peptide was also present in naturally processed peptide extracts of HLA-B7, although its abundance was only 25% of the SMCY peptide abundance (17). Therefore the peptide epitope representing the HLA-B7-restricted HY antigen is de-rived from the protein encoded by SMCY. The location of the SMCY gene and the control of its expression fit well with those expected of the HY antigen based on pre-vious work. Expression of SMCY has been detected in all male tissues tested, as has HY (4, 7, 19). Deletion mapping in humans has placed the HY locus in a portion of interval 6 on the long arm of the human Y chromosome (22), and SMCY maps to this same interval (20). Our work also establish-es that the HY structural gene is encoded on the Y chromosome, rather than being an autosomal gene controlled by Y. The SMCY and SMCX proteins are 85% identical, and the SMCX gene is expressed from both the active and the inactive X chromosomes in both mice and humans (19, 23). Therefore, self-tolerance to SMCX will limit the num-ber of SMCY peptides that could give rise to HY epitopes in association with different MHC molecules. On the other hand, SMCY contains almost 1500 residues, and the over 200 amino acid sequence differences be-tween it and SMCX are scattered relatively uniformly throughout its length. Thus, a large number of distinct SMCY-specific peptides could be generated as HY epitopes. Whether the HY epitope peptides presented by other MHC molecules are also from SMCY is unknown, becausegenetic mapping of the mouse Y chromosome has suggested between two and five distinct loci encoding HY antigens (24). However, a murine HY epitope restricted by H-2Kk has also been shown to be derived from the murine Smcy protein (25). The demonstra-tion that two HY epitopes fromeither mouse or human are derived from the same protein makes SMCY the prime target in searching other HY epitopes. The identification of the protein that gives rise to an HY antigen culminates 40 years of uncertainty regarding its origin and many attempts to identify it. The 77% DNA sequenceidentity betweenSMCY and SMCX provides a likely explanation for past failures to identify HY-encoding genes by subtractive hybridization. Both proteins share significant sequence homology to retinoblastoma binding protein 2, which has been suggested to be a transcrip-