The polymorphic minor histocompatibility Ag HA-1 locus encodes two peptides, HA-1 H and HA-1 R, with a single amino acid difference. Whereas the immunogenicity of the HA-1 R allele has not yet been shown, the nonameric HA-1 H peptide induces HLA-A2-restricted cytotoxic T cells in vivo and in vitro. It is not known whether the mHag HA-1 H or HA-1 R 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-1 H/R peptides for binding to nine HLA class I molecules with> 10% prevalence in the Caucasian population and for seven nonameric/decameric HA-1 H/R peptides predicted to bind to HLA-A3,-B14, and-B60. Only the nonameric KECVL H/R DDL and decameric KECVL H/R DDLL 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-1 H and HA-1 R peptides. In subsequent analyses, dendritic cells pulsed with the nonameric HA-1 R peptide did not induce CTLs that recognize the natural HLA-B60/HA-1 R ligand. In contrast, dendritic cells pulsed with the nonameric HA-1 H peptide induced IFN-γ-secreting T cells specific for the natural HLA-B60/HA-1 H ligand in three HLA-B60+ HA-1 RR individuals, demonstrating the immunogenicity of the HLA-B60/HA-1 H ligand. In conclusion, this study shows a novel HLA-B60-restricted T cell epitope of the minor histocompatibility Ag HA-1 locus.