The repeated injection of low doses of bacterial superantigens (SAg) is known to induce specific T cell unresponsiveness. We show in this study that the spleen of BALB/c mice receiving chronically, staphylococcal enterotoxin B (SEB) contains SEB-specific CD4+ TCRBV8+ T cells exerting an immune regulatory function on SEB-specific primary T cell responses. Suppression affects IL-2 and IFN-γ secretion as well as proliferation of T cells. However, the suppressor cells differ from the natural CD4+ T regulatory cells, described recently in human and mouse, because they do not express cell surface CD25. They are CD152 (CTLA-4)-negative and their regulatory activity is not associated with expression of the NF Foxp3. By contrast, after repeated SEB injection, CD4+ CD25+ splenocytes were heterogenous and contained both effector as well as regulatory cells. In vivo, CD4+ CD25− T regulatory cells prevented SEB-induced death independently of CD4+ CD25+ T cells. Nevertheless, SEB-induced tolerance could not be achieved in thymectomized CD25+ cell-depleted mice because repeated injection of SEB did not avert lethal toxic shock in these animals. Collectively, these data demonstrate that, whereas CD4+ CD25+ T regulatory cells are required for the induction of SAg-induced tolerance, CD4+ CD25− T cells exert their regulatory activity at the maintenance stage of SAg-specific unresponsiveness.