The mammalian Y chromosome encodes male-specific minor histocompatibility (HY) Ags that are recognized by female T cells in an MHC-restricted manner. Two human HY epitopes presented by HLA-A2 and HLA-B7, respectively, have been identified previously and both are derived from the SMCY gene. We previously isolated CD8+ CTL clones that recognized a male-specific minor histocompatibility Ag presented by HLA-B8. In contrast to the SMCY-encoded HY epitopes, the B8/HY Ag was not presented by fibroblasts from male donors, suggesting that it was encoded by a novel gene. We now report that the HLA-B8-restricted HY epitope is defined by the octameric peptide LPHNHTDL corresponding to aa residues 566–573 of the human UTY protein. Transcription of the UTY gene is detected in a wide range of human tissues, but presentation of the UTY-derived HY epitope to CTL by cultured human cells shows significant cell-type specificity. Identification of this CTL-defined HY epitope should facilitate analysis of its contribution to graft/host interactions following sex-mismatched organ and bone marrow transplantation.