Human intraepithelial lymphocytes are T cells primarily of the CD8⁺ phenotype located between intestinal epithelial cells. The cytotoxic and suppressor activities of these lymphocytes are largely unexplored. The spontaneous cytotoxic activity of these cells is evaluated in this study. Jejunal intraepithelial lymphocytes spontaneously lysed a variety of epithelial cell tumor lines (colonic and pancreatic adenocarcinomas and bladder epidermoid carcinoma) but not the highly natural killer-sensitive K-562 cells. Cold target inhibition studies showed that these lymphocytes preferentially bind the DLD-1 colonic adenocarcinoma cells rather than the K-562 cells. Pretreatment of the effector cells with interferon-γ did not change their cytotoxic activity. The cytotoxic cells are T lymphocytes (expressing CD2, CD3, and CD8). In contrast, the spontaneous cytotoxic activity of peripheral blood lymphocytes is directed against both epithelial cell targets and K-562 cells, is enhanced by interferon-γ, and is effected by natural killer cells (expressing CD2, CD16, and NKH1). Thus, the spontaneous cytotoxicity of intraepithelial lymphocytes differs from that of peripheral blood lymphocytes in their target cell restriction, lack of response to interferon gamma, and effector cell phenotype. Lymphocytes in the intestinal epithelium may have a novel and important role in recognizing and destroying transformed epithelial cells and colon cancers.