The present study has demonstrated that suspensions of human peripheral blood lymphocytes can be activated by Con A to become cytotoxic effector cells displaying specificity against a broad range of alloantigen-bearing lymphoid targets. The immunologic nature of the reaction was demonstrated by the fact that autologous targets were not lysed except when an agglutinin (PHA) was present during the cytotoxicity assay. Certain culture conditions were critical for optimal responses including the density of cells, the presence of 2-mercaptoethanol in the culture media, and certain batches of human AB serum which strongly supported the generation of cytotoxic lymphocytes. Cytotoxic cells were generated only after 2 to 3 days of culture and were not present at 24 hr or after 4 days of culture. The optimal concentration of Con A in the culture media was 10 to 20 µg/ml. The target cells employed were human lymphocytes maintained in culture without stimulation for 2 to 3 days. The cytotoxicity assay was a 4-hr assay and relatively high effector-to-target cell ratios (250 to 500:1) were necessary to obtain maximal killing against allogeneic lymphoid targets, whereas lower ratios were adequate against nonlymphoid targets such as Chang liver cells. Con A-generated lymphocytes from particular individuals were consistent in their capacity to mount a high or low cytotoxic response against allogeneic targets. This was independent of the degree of HL-A haplotype similarity or dissimilarity with the target cell. In addition, individuals who were low responders in the Con A-generated cytotoxicity mounted MLC blastogenic responses and MLC-generated alloantigen specific cytotoxic responses as high as those individuals who were high responders in the Con A-generated cytotoxicity assay. Also, lymphocytes from both high and low responders manifested similar degrees of blastogenic responses to Con A. The present study provides a simple and reproducible system for the generation and assay of cytotoxic effector cells from human peripheral blood after polyclonal activation by Con A and provides a model which can be used in studies of the mechanisms and regulatory factors involved in the activation of human cytotoxic lymphocytes.