The immune responses of allogeneic mixed spleen cell cultures (MLC) to the T-dependent antigen, SRBC, and to the T-independent antigen, DNP-PAA, were investigated. The immune response to DNP-PAA in MLC with certain strain combinations was always suppressed as compared with the expected PFC response calculated from the PFC responses of the individual strains. This suppression was eliminated by treating the spleen cells with RAMB antiserum plus complement before the incubation of the MLC with DNP-PAA. It can be concluded that the suppression in the PFC response to the T-independent antigen DNP-PAA in MLC is due to the generation of suppressor T-cells. The PFC response to the T-dependent antigen, SRBC, in MLC showed either suppression, no change, or rarely augmenation, suggesting that the allogeneic mixed spleen cell cultures can generate both suppressor and helper T cells and that the balance between helper and suppressor activity regulates the PFC response to a T-dependent antigen. Suppressor activity was also generated in a one-way MLC, but the degree of suppression depended upon which of the two strains was responding. Similar amounts of thymidine were incorporated in the one-way MLR irrespective of which strains was responding. Thus, the extent of proliferation in one-way MLR is not related to the degree of suppressor activity generated. The results further indicate that a difference between two strains in the I-C, S, and G regions of the major histocompatibility complex is required to generate suppressor activitiy that can depress the response to a T-independent antigen, MLC between strains differing in K, I-A, I-B, I-J, I-E, and D regions generate little or no suppressor activity in this system.