Viable and immunologically competent lymphocytes from unsensitized donors damage allogeneic tissue culture cells in the presence of phytohemagglutinin (PHA). This cytotoxicity is specific since syngeneic tissue culture cells are not at all or only slightly damaged under similar experimental conditions. In this investigation, the relation between the stimulation of human lymphocytes and their cytotoxicity was studied. Chang cells (human liver) served as target cells in all experiments. Cell damage was quantitated by measuring the release of isotope from target cells labeled with chromate-(51)Cr. The cytotoxicity of the lymphocytes was dependent on the concentration of PHA in the incubation medium. Cell damage was maximal at concentrations of 4-8 microl PHA/ml. Higher concentrations were inhibitory although aggregation was increased and no injury of the lymphocytes was noted. Stimulation of DNA and RNA synthesis in PHA-treated lymphocytes each followed dose response curves which were similar to that of cytotoxicity. In order to establish whether stimulation without mixed aggregation of lymphocytes and target cells would suffice for cytotoxicity, a series of nonagglutinating stimulants were investigated. Lymphocytes pretreated with a crude filtrate of Staphylococcus aureus for periods of 0.5-72 hr damaged Chang cells even in the absence of PHA. Lymphocytes from a tuberculin-positive donor were strongly cytotoxic after prestimulation with PPD while those from a negative donor were inactive. Moreover, strong cytotoxic effects were also obtained with lymphocytes which had been stimulated by preincubation with allogeneic lymphocytes in mixed culture. When two stimulants were applied at the same time, additive cytotoxic effects were seen. Addition of PHA to the lymphocyte/Chang cell mixtures potentiated the cytotoxicity of prestimulated lymphocytes. The cytotoxic potential of the lymphocytes was in all cases correlated to the degree of stimulation recorded as transformation into blast cells, and was independent both of the degree of aggregation and of the stimulating factor. These findings are compatible with the assumption that injury of the Chang cells reflected an immunologically nonspecific activity of lymphocytes enhanced by stimulation. The possible importance of this activity for a number of tissue-damaging immune reactions in vivo is pointed out.