Tyrosinase-related protein 2 (TRP2) is a melanosomal enzyme expressed in most mammalian melanocytes and melanomas. This protein has been identified as a melanoma antigen recognized by tumor reactive CTLs derived from tumor infiltrating lymphocytes in the context of HLA-A31 and HLA-A33. The frequencies of these HLA-A alleles among melanoma patients in the United States is low (∼6% for HLA-A31 and ∼2% for HLA-A33) compared with that of HLA-A*0201 (∼46%). Therefore, to extend significantly the use of TRP2-based immunotherapies for the treatment of patients with melanoma, we searched for new HLA-A*0201-restricted epitopes from this protein by screening TRP2-derived peptides for the induction of melanoma-reactive CTL. Fifty-one peptides were selected from TRP2 based on a permissive HLA-A*0201 binding motif, and the 21 peptides with the highest experimentally determined binding affinities were used to stimulate peripheral blood lymphocytes from HLA-A*0201⁺ melanoma patients in vitro. One peptide, TRP2(180–188) (SVYDFFVWL), induced CTLs from three of four patients that specifically recognized peptide-pulsed T2 cells, COS-7 cells expressing HLA-A*0201 and TRP2, and HLA-A2⁺ TRP2⁺ melanomas. TRP2(180–188) is identical to a previously identified TRP2 epitope recognized by murine melanoma-reactive CTLs in the context of H-2K^b. These results suggest that TRP2 may be useful for the development of murine tumor immunotherapy models and for the treatment of melanoma patients who are diverse in HLA expression.