In the present work, to analyze the heterogeneity of the tumor-specific cytotoxic immune response, a large number of T cell clones were generated from the infiltrate of a tumor-proximal invaded lymph node, and two kinds of melanoma-specific CD8+ CTL clones were derived. The majority of T cell clones (about a hundred) are characterized by a specific lysis of the autologous tumor cell lines. Among 34 of the latter clones, HLA-A2 molecule and MART-1(27-35) peptide have been shown to play a predominant role in tumor recognition. However, no significant amplification at the tumor site was observed for 3 of these CTL. The other kind of tumor-specific CTL (1 oligoclonal and 2 clonal cell lines) did not lyse the autologous melanoma cell lines but lysed the "fresh" autologous tumor cells in a MHC class I-dependent manner. Functional analysis of the two different CTL clones have shown that they did not lyse NK targets, autologous peripheral monocytes, activated T cells, and transformed B cells or any of the few allogeneic cultured and uncultured melanoma cells we tested. TCR repertoire analysis has shown that one of these CTL clones was significantly detectable "in situ" among tumor-infiltrating lymphocytes, while not detectable among PBMC. Such melanoma-specific lymphocytes, which could not have been picked out through conventional screening procedures using tumor cell lines, could potentially play a role in tumor rejection. These results suggest that the immune response analyzed toward melanoma cell lines does not totally reflect the in situ immune status.