Long‐term growth of tumor‐infiltrating lymphocytes (TIL) in high concentrations of rlL‐2 is required for generation of therapeutic numbers of cells for adoptive immunotherapy of human cancer. Under these conditions rlL‐2 promotes both anti‐tumor cytotoxicity and lymphocyte growth from tumors of several histological types. In a series of 16 consecutive tumors, studies of TIL‐mediated cytotoxicity against different tumor targets were characterized by an initial strong tumor‐non‐specific cytotoxicity. With time, TIL bulk cultures became non‐cytotoxic against all targets (median time = 38 days). Non‐cytotoxic TIL bulk populations were capable of mediating strong cytotoxic responses if pre‐treated with anti‐T‐cell antigen receptor antibody (TcR) before addition to targets. TIL populations were not, however, uniformly susceptible to anti‐TcR‐mediated cytotoxicity. Anti‐TcR‐mediated cytotoxicity was confined to CD8+ bulk populations (defined as populations with a CD4/CD8 ratio <l), with virtually no cytotoxicity observed in CD4+ populations (CD4/CD8 ratio >2), (p < 0.01). Both CD4 and CD8 populations expressed TcR antigen reactive with anti‐TcR antibody. These results indicate that, despite poor in vitro anti‐tumor cytotoxicity in short‐term assays, CD8+ TIL are fully competent cytotoxic effector cells when subjected to strong activation signals via the TcR complex. In addition, these results imply that adoptively transferred CD4+ populations of TIL have in vivo biologic functions quite distinct from those of CD8+ populations and, further, that disparate clinical outcomes could reasonably be expected from the adoptive transfer of either population alone.