The detection of T cells with specificity for human renal cell carcinoma (RCC) has been difficult to document. In an attempt to improve our identification of RCC-reactive T cells, tumor-infiltrating lymphocytes (TIL) were expanded in interleukin-2/interleukin-4 (IL-2/IL-4) and then separated into CD4+ and CD8+ subsets using antibody-coated biomagnetic beads. TIL grown in IL-2/IL-4 expanded to greater numbers than TIL grown in IL-2 alone. From 16 patients in whom subset separation was performed, three CD4+ and three CD8+ TIL consistently had specificity for RCC that was detected by cytotoxicity, proliferation, or interferon-gamma (IFN-gamma) production. Four of the six lines were derived from the IL-2/IL-4 cultures. Two CD8+ TIL lines displayed specific lytic activity, lysing the autologous tumor but not allogeneic RCC or nonrenal tumors. Moreover, the lytic activity of these lines was blocked by anti-CD3 antibody, suggesting that tumor recognition was through the TCR/CD3 complex. Two additional TIL lines showed preferential lysis of RCC because they were cytotoxic for autologous tumor and one or more allogeneic RCC but not other tumor types. Two nonlytic CD4+ lines as well as the two CD8+ lines that were specifically lytic also produced IFN-gamma in response to the autologous tumor but not allogeneic RCC. Although these TIL lines produce IFN-gamma when stimulated with tumor alone, the addition of 5 U/ml of IL-2 significantly enhanced IFN-gamma secretion. The four TIL lines that showed specificity for RCC in terms of IFN-gamma production also had enhanced proliferation to the autologous RCC plus IL-2 but not to multiple allogeneic RCC plus IL-2. These studies demonstrate that TIL from RCC patients contain both CD4+ and CD8+ T cells that have specificity for RCC. In addition to cytotoxicity, specificity to RCC can be defined by IFN-gamma production and proliferation.