Graft rejection after histocompatibility locus antigen (HLA)-identical stem cell transplantation results from the recognition of minor histocompatibility antigens on donor stem cells by immunocompetent T lymphocytes of recipient origin. T-lymphocyte clones that specifically recognize H-Y epitopes on male target cells have been generated during graft rejection after sex-mismatched transplantation. Previously, 2 human H-Y epitopes derived from the same SMCY gene have been identified that were involved in bone marrow graft rejection. We report the identification of a new male-specific transplantation antigen encoded by the Y-chromosome-specific gene DFFRY. The DFFRY-derived peptide was recognized by an HLA-A1 restricted CTL clone, generated during graft rejection from a female patient with acute myeloid leukemia who rejected HLA-phenotypically identical bone marrow from her father. The identification of this gene demonstrates that at least 2 genes present on the human Y-chromosome code for male-specific transplantation antigens.