We have previously described the generation of tumor-infiltrating lymphocyte (TIL) clones from renal cell cancer by solid-phase anti-CD3 antibody activation and expansion in 100 IU/ml IL-2 plus irradiated allogeneic B cells. These culture conditions did not select for a particular T cell subset. Using these culture conditions, we report here the generation of 66 CD4+ and 36 CD8+ TIL clones from five patients with melanoma. Eighty-five percent of the CD4+ TIL clones were not cytolytic (< 30% lysis, E:T 20:1) as determined by antibody-redirected lysis (ARL), whereas all CD8+ clones showed strong ARL activity (> 30% lysis). Clones were further tested for production of IL-2, IL-4, and IFN-gamma after activation for 48 hr by solid-phase anti-CD3. CD8+ clones produced significant amounts of IFN-gamma, little IL-2, and no IL-4. CD4+ clones were classified as Th0, Th1, or Th2, analogous to the classification of T helper cells in the mouse. Sixty-six percent existed as Th0, producing IL-2, IL-4, and IFN-gamma. Only 15% existed as Th1, producing IL-2 and IFN-gamma, and 19% as Th2, producing IL-4 but no IL-2 or IFN-gamma. In all cases, unstimulated clones or clones stimulated with the allogeneic B cell line did not produce detectable amounts of cytokines. Solid-phase anti-CD3 activation was compared to activation with autologous melanoma cells. Five of nine CD8+ clones produced low amounts of IL-2 (< 200 pg/ml/10(6) cells) in response to autologous tumor, but none of the CD8 clones produced IFN-gamma or IL-4. Also, 5/7 Th0 clones from one patient produced similar amounts of IL-2 after stimulation with anti-CD3 or autologous tumor. The other two clones produced only 10% or less of the amount produced in response to anti-CD3. No IL-4 or IFN-gamma could be detected in response to autologous tumor. In contrast, none of the 12 T helper clones from two other patients produced any cytokines after stimulation with autologous tumor cells. Together these data suggest that the T cell infiltrate in melanoma consists primarily of IL-2-producing Th0 cells, but few of those are triggered by autologous tumor cells.