Coculture of spleen cells from mice tolerized with 2,4-dinitrobenzenesulfonate (DNBS) and DNP-labeled spleen cells (DNP-SC) activates Lyt-2+ T suppressor cells (Ts) to synthesize and release a suppressor factor (SSF) into the supernatant, which suppresses the transfer of contact sensitivity to DNFB. The purpose of the present study was to examine in greater detail the signals required to activate DNBS-primed Ts to produce SSF. The supernatant from cultures of tolerant cells and glutaraldehyde-fixed DNP-SC did not have SSF. In contrast, the soluble cell lysate from these cultures did contain the suppressive activity. Pretreatment of glutaraldehyde-fixed DNP-SC with either anti-DNP or anti-class I, but not anti-class II MHC, antibodies blocked SSF synthesis. The addition of IL 1 to cultures of DNBS-tolerant cells and glutaraldehyde fixed DNP-SC restored the ability of the Ts to release the synthesized factor. These results indicate that Ts recognition of the hapten/class I MHC determinant stimulates the synthesis of SSF, and a costimulator is required to induce the release of the factor. The supernatants from cultures of either L3T4-depleted tolerant cells and DNP-SC or tolerant cells and anti-I-A antibody-treated DNP-SC had no SSF activity. The addition of a costimulator (IL 1) also restored the ability of the Ts to release the synthesized factor in cultures of L3T4-depleted tolerant cells and DNP-SC. These results suggest that an L3T4 cell in the DNBS-primed cell population interacts with I-A determinants on a cell in the DNP-stimulator population to initiate the generation of the mediator required for SSF release. This further suggests that the Ts is unable to induce the costimulator from the hapten-presenting cell during interaction with the DNP/class I MHC ligand. Therefore, the production of SSF is regulated not only by the presentation of the appropriate hapten/MHC determinant but also by the interactions of cells that function in generating the costimulator needed to induce release of the suppressor factor.