A short term microcytotoxicity assay system using radioisotope release as an index of target cell damage has been developed to evaluate immune killing of cultured adherent human colon cancer cell lines. Using this assay system, complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and spontaneous cell-mediated cytoxicity of adherent human colon cancer cell lines can be assessed in 4 hr or less. An essential step in the development of this system was the successful 51Cr-labeling of human colon cancer target cells with subsequent low spontaneous release. This was achieved through careful attention to cellular growth phase and medium pH during the labeling and assay period. In this microsystem, labeled colon cancer cells spontaneously released 51Cr at a mean rate of 2% per hr during the assay, a level low enough not to obscure specific cytotoxic responses. Complement-dependent cytoxicity was measured most conveniently over a 2-hr period, whereas antibody-dependent cell-mediated cytotoxicity and spontaneous cell-mediated cytotoxicity were optimal when measured over a 4-hr period.