We characterized a new melanoma antigen derived from one of the multiple open reading frames (ORFs) of the meloe transcript. The meloe gene is overexpressed in melanomas as compared to other cancer cell lines and normal tissues. The corresponding transcript is rather unusual, in that it does not contain a long unique ORF but multiple short ORFs. We recently characterized a tumor epitope derived from a polypeptide (MELOE-1) encoded by the ORF(1230-1370) and involved in relapse prevention of melanoma patients treated with autologous tumor infiltrating lymphocytes (TIL). Here we show that the ORF(285-404) encodes a polypeptide called MELOE-2 that also generated a HLA-A2 epitope recognized by a melanoma-specific T cell clone derived from the same TIL population from which we derived the MELOE-1-specific T cell clone. We also showed that HLA-A2 melanoma cells were spontaneously recognized by the MELOE-2-specific T cell clone, and we detected the presence of MELOE-2 reactive T cells in another TIL population infused to a patient who remained relapse-free after TIL treatment. These results demonstrate that translation of meloe transcript in melanoma cells can produce at least two immunogenic polypeptides, MELOE-1 and MELOE-2, from two distinct ORFs that could be relevant target for melanoma immunotherapy.