Lymphocytes infiltrating human solid tumors (TIL) and autologous peripheral blood lymphocytes (A-PBL) were cultured with 1000 units/ml of recombinant interleukin 2 (rIL2) in long-term cultures. TIL isolated from 26 primary squamous cell carcinomas of the head and neck expanded better (P less than 0.01) and achieved higher total lytic units of activity against fresh tumor cell targets (P less than 0.05) than A-PBL. TIL obtained from primary hepatocellular carcinomas (n = 7) showed a higher degree of expansion than those from metastatic liver tumors (n = 7). Further, TIL from metastatic tumors of the head and neck, liver, and ovary were delayed up to 50 days in their proliferative response to rIL2. Long-term mass cultures in rIL2 of TIL, A-PBL, or normal PBL were serially monitored for cytotoxicity with different cultured and fresh tumor cell targets and for phenotypic markers of the predominating cell populations. Antitumor cytotoxicity was found in cultures enriched in CD3+Leu19+ and/or CD3-Leu19+ cells. Two-color sorting of such cultures followed by cytotoxicity assays confirmed that the human antitumor effectors expressed either the CD3+Leu19+ or CD3-Leu19+ phenotype. CD3+Leu19- cells had little or no antitumor cytotoxicity. The two types of Leu19+ effector cells were present in low numbers in fresh TIL, A-PBL, or normal PBL; in contrast, in some rIL2-expanded long-term cultures, they represented a majority of proliferating cells. This study identifies for the first time two types of antitumor effector cells in rIL2 cultures of human TIL, one of which may represent activated natural killer cells on the basis of the absence of the CD3 and expression of the Leu19 antigen. These antitumor effector cells mediate non-MHC-restricted cytotoxicity of fresh or cultured tumor cell targets of different histologic types.