In normal mice, the autologous mixed lymphocyte reaction (AMLR) can activate helper T cells that, in the presence of hapten-modified syngeneic cells, can induce a hapten-specific cytotoxic response. Supernatants from AMLR cultures contain a factor(s) that will mediate a cytotoxic T cell response to hapten-altered self. The AMLR factor is effective in facilitating the generation of cytotoxicity only in those cultures containing both T cells and hapten-altered, syngeneic, nonstimulatory cells. Factor production requires an interaction between Lyt-1+23- cells and non-T cells (the T cells synthesize it). The AMLR factor does not appear to be interleukin 2 (IL-2) because it does not activate thymocytes in the presence of antigen, nor does it maintain an IL-2-dependent cell line or function in co-stimulator assays. For the AMLR factor to facilitate the generation of cytotoxicity, thymic adherent cells are a necessary intermediate. These data suggest that the factor recoverable from AMLR cultures acts early in the cytotoxic pathway, before IL-1 production.