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It is well established that the immune response to alloantigens is characterized by the formation of effector cells which specifically destroy target cells carrying the sensitizing alloantigens in the absence of antibody and/or complement (for references, see 1). These effector cells belong to the thymus-derived (T) lymphocyte series and are generally referred to as cytotoxic T lymphocytes (CTL)1With the development of a precise and reproducible cytotoxic assay system (2), it has been possible to follow the appearance of CTL during the primary and secondary response of mice to normal or tumor allografts. In certain instances, such studies have shown an earlier appearance and a higher peak of cytotoxic activity in spleens of mice injected twice with allogeneic tumor cells as compared to that observed after primary immunization, suggesting the existence of an anamnestic response in T-cell-mediated immunity (3-4).Recently, it has been found that mouse CTL can be generated in vitro using mixed leukocyte cultures (MLC). Following the work of H~yry and Defendi (5), several studies have established the usefulness of the MLC system in providing an in vitro experimental model to analyze the series of events resulting in the appearance of CTL (6-10).The present study was undertaken to investigate the effect of in vivo immunization on CTL generation in MLC. Using improved tissue culture conditions, we found that cell-mediated cytotoxic responses were significantly higher when the responding cells were obtained from immune (MLC-Imm) rather than normal spleens. At the peak of the response, MLC-Imm populations were five times more cytotoxic than MLC populations. Physical and immunological characterization of the effector cells generated in MLC-Imm gave results simi-

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