Tumor-infiltrating lymphocytes (TIL) were isolated by enzymatic digestion and gradient centrifugation from 18 human ovarian carcinomas. These cells were cultured in a complete medium supplemented with recombinant interleukin 2 (IL2) alone or recombinant IL2 plus recombinant tumor necrosis factor α (TNF-α), and their growth and antitumor cytotoxicity were determined. TIL cultured in the presence of IL2 plus TNF-α (1000 units/ml each) for 6 days showed significantly higher cytotoxicity against fresh autologous tumor targets than did TIL cultured with IL2 alone (e.g., mean lytic units/10⁷ cells for 8 TIL preparations were 290 versus 74; P < 0.05). No differences in [³H]thymidine uptake or natural killer cell activity were observed among these TIL cultures. In titration experiments, optimal synergistic concentrations of IL2 and TNF-α were determined as 10² and 10³ units/ml, respectively. Using these concentrations for culturing the TIL, effector cells developed which preferentially lysed autologous tumor and displayed a CD8⁺ phenotype (up to 75% positive). However, the autologous tumor cytotoxicity mediated by these cultured TIL on day 6 was short lived. By day 12, it was replaced by non-major histocompatibility complex-restricted, lymphokine-actived killer cell-like activity mediated by CD3^-CD56⁺ effector cells. Simultaneously, the production of γ-interferon and interleukin 1 decreased in these cultures. In contrast to TNF-α, anti-CD3 antibody synergized with IL2 to increase 2–3-fold TIL proliferation but not their cytotoxic activity against autologous tumor cell targets. These data suggest that TNF-α and IL2 synergize early in culture to induce tumor-reactive CD8⁺ effectors, some of which may be specific for autologous ovarian tumor cells. However, the conditions needed to sustain the specific autologous tumor responses in long-term cultures of human TIL remain to be determined.