It was previously found that primary melenomas from HLA-A2-matched patients display an increased expression of a few T cell receptor (TCR) variable-region beta-chain transcripts (BV) compared with autologous peripheral blood lymphocytes (PBL) and uninvolved skin. In order to see whether expansions of clonal/oligoclonal subsets of T cells occurred, complementarity-determining region 3 (CDR3) of BV transcripts overexpressed in the neoplastic infiltrate were cloned and sequenced. Dominant rearrangements were found for BV14, which were commonly increased in the neoplastic lesions of all analysed HLA-A2 melanoma patients, as well as for other overexpressed BV gene families, but none of them could be identified among autologous PBL. No identical CDR3 sequences could be detected in the dominant BV14 rearrangements obtained from the different patients. In one patient a single clonally expanded SLSGTGVNEQF CDR3 clonotype accounted for the entire BV14 relative frequency of expression (24%) in tumor-infiltrating lymphocytes (TIL). Two independent mixed lymphocyte/tumor cultures (MLTC) could be successfully established from TIL of the patient and were found to exert HLA-class-I-restricted cytotoxicity for the autologous melanoma line. BV14 T cells that constituted from 22% to 32% of all T cells present in both MLTC lines, as assessed by flow cytometry, all displayed the same CDR3 clonotype found in vivo and could be shown, by TCR down-modulation experiments, to be involved in autologous tumor recognition. These results support the hypothesis of a tumor-antigen-driven origin of clonally amplified T cells present at high frequency in the in situ neoplastic infiltrate.