This study demonstrated that antisuppressor T cells (TAS) were generated in allogeneic mixed lymphocyte culture (MLC) when supplemented with a conditioned medium containing T cell differentiation factor (CM-TCDF). CM-TCDF was shown to efficiently restore the alloreactive cytotoxic T lymphocytes (CTL) response in accessory cell-depleted MLC. CM-TCDF could also sustain the growth and cytotoxic activity of bulk-cultured CTL. In contrast, cloned CTL only required interleukin 2 to maintain their growth and cytotoxic activity. These findings suggested that bulk-cultured MLC might contain different populations of immunoregulatory cells in addition to CTL effectors. These immunoregulatory cells provide "on" or "off" signals to turn on or turn off the lytic machinery of CTL. The suppressor T cells (Ts) generated in MLC might provide the "off" signal to CTL that resulted in the termination of their cytotoxic activity after 7 days of culturing. When CM-TCDF was supplemented in MLC, we found that TAS were generated. TAS could efficiently abrogate the suppressor activity of anti-allo-TS, but they had no effect on the anti-self veto cell activity. Both the TAS and TS activities were allospecific. The precursors and effectors of TAS were both found to be L3T4+ cells, whereas the TS effectors were Lyt-2+ cells. Generation of TAS was completely blocked by alpha L3T4 antibody and was partially blocked by alpha Thy-1 antibody. In contrast, alpha Lyt-2 antibody or antibodies against class II major histocompatibility complex framework determinants had no effect on TAS generation. Therefore, TAS were different from L3T4+ T helper cells that were induced in the context of recognizing class II major histocompatibility complex determinants. TAS might represent a separate set of immunoregulatory cells that provide an "off" signal to the TS, which allowed the lytic machinery of the CTL to remain active and, thus, to maintain their cytotoxic activity for a prolonged period of time.