The specificity of cytotoxic T lymphocyte (CTL) responses generated in the presence of lymphokines was studied. Thymic responder cells were activated in the presence of stimulator cells that differed in their metabolic activity. After 5 days of culture, the cytotoxic response was estimated in a 4-h 51Cr-release test. Coculture of thymic responders with irradiated splenic stimulator cells in the presence of interleukin 2(IL 2) led to preferential cytolysis of target cells that expressed the same histocompatibility antigens as the cells used for sensitization. Addition of T cell cytotoxicity-inducing factor 1 (TCF1), however, to those cultures made the presence of stimulator cells unnecessary and induced cytotoxic responses against all target cells tested, including target cells syngeneic to the responder cells. This activation was neither due to contaminating mitogen nor to the effect of heterologous serum in the assay system. The conclusion of these findings was that either polyclonal activation of CTL was induced by TCF1 or that some specific CTL clones differentiated into unrestricted killer cells under the influence of TCF1.