Tumor-infiltrating lymphocytes and autologous peripheral blood lymphocytes from 7 patients with adenocarcinoma of the colon were evaluated for expansion and antitumor activities during culture in the presence of 1000 U/ml of recombinant human interleukin 2. Functional and phenotypic characteristics of tumor-infiltrating lymphocytes and autologous peripheral blood lymphocytes were compared between weeks 2 and 3 of culture in recombinant interleukin 2. All but one tumor-infiltrating lymphocyte and all autologous peripheral blood lymphocyte preparations proliferated well in vitro. Tumor-infiltrating lymphocytes expanded better (p less than 0.05) than autologous peripheral blood lymphocytes, reaching median-fold expansions of 2231 (range 1-4720) and 108 (range 13-1263), respectively. Cytotoxicity of interleukin 2-activated tumor-infiltrating lymphocytes and autologous peripheral blood lymphocytes against fresh autologous or allogeneic colon carcinoma targets was relatively low in these cultures. Due to better proliferation, tumor-infiltrating lymphocytes showed significantly greater (p less than 0.05) total cytotoxic activity per culture against fresh autologous tumor-cell targets than did autologous peripheral blood lymphocytes, achieving a median total lytic units of activity per culture of 671 compared with 92 for autologous peripheral blood lymphocytes. Cytotoxicity was not restricted to autologous tumor cells. Two-color flow cytometry demonstrated that the predominant proliferating cell population in interleukin 2-expanded long-term cultures of tumor-infiltrating lymphocytes and autologous peripheral blood lymphocytes expressed the CD3+Leu19- phenotype. Some cultures were enriched in CD3+ Leu19+ and CD3-Leu19+ cells. This study indicated that tumor-infiltrating lymphocytes from most but not all human primary colon adenocarcinomas could be expanded in the presence of recombinant interleukin 2 and mediate non-major histocompatibility complex-restricted antitumor cytotoxicity. Because fresh colon carcinoma cells appear to be resistant to in vitro lysis by interleukin 2-activated tumor-infiltrating lymphocyte and autologous peripheral blood lymphocyte effectors, the role of adoptive immuno-therapy in treatment of advanced colon carcinomas in humans may have to be reevaluated.