The polymorphic minor histocompatibility Ag HA-1 locus encodes two peptides, HA-1H and HA-1R, with a single amino acid difference. Whereas the immunogenicity of the HA-1R allele has not yet been shown, the nonameric HA-1H peptide induces HLA-A2-restricted cytotoxic T cells in vivo and in vitro. It is not known whether the mHag HA-1H or HA-1R associates with other HLA class I molecules. Therefore, the polymorphic regions of both HA-1 alleles were analyzed to identify HLA class I binding peptides that are properly processed by proteasomal degradation. Peptide binding analyses were performed for all nonameric HA-1H/R peptides for binding to nine HLA class I molecules with >10% prevalence in the Caucasian population and for seven nonameric/decameric HA-1H/R peptides predicted to bind to HLA-A3, -B14, and -B60 Only the nonameric KECVLH/RDDL and decameric KECVLH/RDDLL peptides showed strong and stable binding to HLA-B60. In vitro digestion of 29-aa-long HA-1 peptides by purified 20S proteasomes revealed proper cleavage at the COOH termini of both HLA-B60 binding HA-1H and HA-1R peptides. In subsequent analyses, dendritic cells pulsed with the nonameric HA-1R peptide did not induce CTLs that recognize the natural HLA-B60/HA 1R ligand. In contrast, dendritic cells pulsed with the nonameric HA-1H peptide induced IFN-τ-secreting T cells specific for the natural HLA-B60/HA-1H ligand in three HLA-B60+HA-1RR individuals, demonstrating the immunogenicity of the HLA-B60/HA-1H ligand. In conclusion, this study shows a novel HLA-B60-restricted T cell epitope of the minor histocompatibility Ag HA-1 locus.