To try to understand the functional significance of human melanoma-infiltrating lymphocytes (TIL), a clonal analysis of the specificity, T cell receptor (TcR) diversity and activation requirements of these lymphocytes isolated from four different tumors was carried out. Supporting the presence of in vivo primed tumor-specific T lymphocytes in these four tumors, a high frequency of the Cd8+ and CD4+ clones, obtained from the TIL cultured for a few days with recombinant interleukin (rIL)-2 and autologous tumor cells, exhibited a restricted lysis or proliferation in response to the autologous tumor cell line. In contrast, no tumor-specific clone was obtained from freshly extracted TIL, suggesting that the frequency of tumor-specific effectors remained low in these tumors. Only the CD8+ clones lysed the autologous tumor cells and their activity was major histocompatibility complex MHC class I restricted. Significant expansion of CD4+ and CD8+ tumor-specific clones required regular restimulation by autologous melanoma cells but also the addition of exogenous IL-2 and of Epstein-Barr virus-transformed B feeder cells. Five different tumor-specific clones, three CD8+ and two CD4+ clones were identified in a single tumor on the basis of their TcR gene configuration. Together, these data suggest that a spontaneous and diverse immune response, mediated by tumor-specific CD4+ as well as CD8+ T lymphocytes, arises in most MHC-bearing human melanomas but that antigen-MHC complex presentation by tumor cells does not, at least in vitro, allow a significant proliferation of these lymphocytes.