Melanoma-associated peptides recognized by cytolytic T lymphocytes (CTL) in the context of several histocompatibility leukocyte antigens (HLA) are required for the development of specific immunotherapies. Using a transient transfection assay into COS-7 cells, we identified the gp100/pMel17 melanosomal protein as the shared antigen recognized by three independent CD8+ CTL clones in HLA-A*6801-restricted fashion. This finding was confirmed by the correlation between lack of gp100/pMel17 protein in a number of HLA-A*6801-positive melanomas and their resistance to lysis/cytokine production by the specific effectors. The gp100/pMel17 antigenic epitope was identified based on recognition of subfragments and on a computer-based prediction algorithm. Among a panel of gp100/pMel17-derived synthetic peptides only the 10-mer HTMEVTVYHR (gp100/pMel17182-191) induced tumor necrosis factor (TNF) release by CTL clones when pulsed on suitable target cells whereas both the 10-mer and the shorter 9-mer gp100/pMel17183-191 sensitized the same antigen-pulsed cells to lysis. In conclusion, the identification of the HTMEVTVYHR peptide will extend to HLA-A*6801 melanoma patients the possibility to exploit gp100/pMel17 melanosomal protein for experimental and clinical studies.