Patients with colorectal cancer respond poorly to in vivo immunotherapy with lymphokine-activated killer (LAK) cells generated from peripheral blood mononuclear cells (PBMC). We postulated that gut-derived immune cells could be a more relevant source of LAK cells directed against colorectal cancer. Intestinal lamina propria mononuclear cells (LPMC) and colonic adenocarcinoma cells were isolated from operative specimens by combination of mechanical and enzymatic dissociation methods. LAK cells were generated by culturing PBMC and LPMC with recombinant interleukin 2 (IL2), with and without OKT3 monoclonal antibody, in short- (4 days) and long-term (21 days) cultures. Other cultured tumor cells, normal intestinal fibroblasts, and hapten-modified autologous LPMC were used as control targets. Cytotoxicity was measured by a 4-hr 51Cr release assay. Short-term cultured LAK cells exhibited a strong to moderate degree of killing against normal intestinal fibroblasts, hapten-modified self cells, and four different tumor cell lines. Instead, fresh colon cancer cells were resistant to cytotoxicity, regardless of their degree of histologic differentiation and the autologous or allogeneic nature of the LAK cells. Long-term culture with IL2 remarkably increased LAK cell activity against all tumor targets, but not against colonic adenocarcinoma cells. The results of this study, showing that freshly isolated colon cancer cells are intrinsically resistant in vitro to highly activated cytotoxic effector cells, may explain the poor clinical results observed in human trials with in vivo administration of IL2 or LAK cells.