The availability of antitumor cytotoxic T lymphocytes which can be generated from either peripheral blood lymphocytes after stimulation in vitro or tumor infiltrating lymphocytes (TIL) has made it possible to identify a number of melanoma antigens presented by major histocompatibility complex class I molecules. The present and previous studies indicated that TIL586 recognized an antigen expressed on most melanoma and normal melanocytes in the context of the HLA-A31 molecule. We report here the cloning of a cDNA that directs the expression of the shared melanoma antigen recognized by this TIL. The DNA sequence analysis revealed that the cDNA was almost identical to the gene encoding tyrosinase-related protein 1 or glycoprotein gp75 which was originally identified by serum antibodies in a patient with melanoma. The gene was found to be expressed only in melanoma, normal melanocyte cell lines, and retina, but not in other normal tissues tested. The gp75 antigen presented by HLA-A31 may therefore constitute a useful immune target for specific treatment of patients with melanoma, since both antibody- and T cell-mediated immune responses can be generated against this antigen.