Cytotoxic T lymphocytes (CTL) were not generated in an autologous mixed lymphocyte reaction (MLR) in the presence of human AB serum. No CTL were generated in cultures that contained T cells and irradiated autologous, allogenetic, or hapten-modified autologous T lymphocytes. However, when allogeneic T lymphocytes or hapten-modified autologous T lymphocytes were added to an autologous MLR, specific CTL were generated. Furthermore, supernatant medium from an autologous MLR allowed the generation of specific CTL in T cell cultures to which allogeneic T cells or hapten-modified autologous T cells were added.