Mouse cytotoxic T lymphocytes (CTL) were generated in mixed leukocyte cultures (MLC) using spleen cells as responding cells and irradiated allogeneic spleen cells as stimulating cells. Cytotoxicity was assessed by a quantitative (51)Cr assay system and the relative frequency of CTL in individual cell populations was estimated from dose-response curves. Inclusion of 2-mercaptoethanol in the MLC medium resulted in a 20-40-fold increase in the relative number of CTL generated at the peak of the response. Under these culture conditions, cell-mediated cytotoxic activity was detectable in MLC populations as early as 48 h after the onset of the cultures. When spleen cells from mice immunized with allogeneic tumor cells 2-4 mo previously were cultured with irradiated spleen cells of the same alloantigenic specificity (MLC-Imm), it was found that the cell-mediated cytotoxic response was detectable earlier and reached higher levels than that observed in a primary MLC. At the peak of the response, MLC-Imm populations were observed to lyse up to 50% of the target cells within 3 h at a lymphocyte: target cell ratio of 0.3:1. Immunological and physical characterization of the effector cells generated in MLC-Imm indicated that they were medium to large-sized T lymphocytes. Altogether, these studies suggested the existence of an anamnestic cell-mediated cytotoxic response in MLC-Imm.