Tumor-reactive CD4+ T cells can be isolated and expanded from the peripheral blood and tumor lesions of patients with melanoma. In contrast to CD8+ T cells, little is known about the antigens recognized by these CD4+ T cells. As a consequence, little is known about the diversity of the T-cell receptor (TcR) use by melanoma-reactive CD4+ T cells. To address these questions, a panel of clonal or highly oligoclonal CD4+ T-cell lines was established from a patient with metastatic melanoma. A CD4+ tumor-infiltrating lymphocyte (TIL) line was established that was highly oligoclonal and recognized only autologous melanoma cells but not allogeneic melanomas, suggesting the expression of a mutated or uniquely expressed antigen by this melanoma. The antigen recognized by the CD4+ TILs could be presented by intact melanoma cells or by autologous Epstein-Barr virus (EBV) B cells pulsed with melanoma cell lysates. A panel of CD4+ clonal and highly oligoclonal T-cell lines was isolated form peripheral blood mononuclear cells (PBMC) from this patient; these were also reactive with autologous melanoma cells or tumor extracts pulsed on autologous EBV B cells. Despite their reactivity with the autologous melanoma, we found no evidence of restricted TcR V gene use, because all six T-cell lines recognized antigen via different TcR alpha/beta rearrangements. Furthermore, there were no conserved amino acids in the CDR3 regions of these TcRs, indicating that multiple TcR clonotypes could mediate recognition of a single unique major histocompatibility (MHC) complex class II restricted melanoma antigen or that multiple MHC class II restricted melanoma antigens are expressed by the melanoma.