The role of adherent cells (MØ) in 1) primary in vitro PFC responses and 2) in vitro generation of suppressor T cells (Ts) to the antigen L-glutamic acid60-L-alanine30-L-tyrosine10 in responder strain mice has been investigated. Removal of MØ from spleen cell suspensions by either serial culture on plastic surfaces or passage over Sephadex G-10 beads yielded lymphocytes unable to give primary in vitro PFC responses under modified Mishell-Dutton conditions to either soluble GAT or the particulate antigens sheep red blood cells (SRBC) or GAT-complexed electrostatically to methylated bovine serum albumin (GAT-MBSA). However, the viability of such MØ-depleted cultures was significantly lower than control cultures of unseparated spleen cells. Addition of 5 × 10-5 M 2-mercaptoethanol (2-ME) to the culture medium restored viability and permitted nonadherent cells to respond to SRBC and GAT-MBSA, but not to soluble GAT. The response of nonadherent cells to soluble GAT could be restored in the absence of 2-ME by the addition of 1 to 3 × 105 x-irradiated, anti-Thy 1.2 + C-treated peritoneal exudate cells (MØ), or in the presence of 2-ME by 1 to 2 × 104 MØ. This low number of MØ was identical to the number of GAT-bearing MØ necessary in the presence of 2-ME to give optimal GAT PFC responses. Taken together, these data indicate a crucial antigen-presenting role for MØ in primary in vitro anti-GAT responses. Removal of such antigen-presenting cells from responder spleen cells left a lymphocyte population, which, when cultured for 2 days with normally immunogenic (10 µg/ml) concentrations of GAT, gave rise to highly active Thy 1.2+ GAT-specific suppressor cells. This Ts induction was demonstrated in several responder strains. The GAT-Ts were unable to suppress the in vitro responses to SRBC or the copolymer L-glutamic acid50-l-tyrosine50 (GT) as GT-MBSA. Increasing the concentration of GAT to ≥ 30 µg/ml permitted the induction of GAT-Ts in unseparated spleen cells. Thus, in responder mice, MØ able to present antigen in an immunogenic form play a central role in regulating the balance of activated TH and Ts, suggesting that an Ir gene defect in nonresponder mice at the MØ level for this presentation function may account for the predominant suppressor T cell responses of such mice.