CD4+ CD25+ regulatory T cells inhibit organ-specific autoimmune diseases induced by CD4+ CD25− T cells and are potent suppressors of CD4+ CD25− T cell activation in vitro. We demonstrate that CD4+ CD25+ T cells also suppress both proliferation and IFN-γ production by CD8+ T cells induced either by polyclonal or Ag-specific stimuli. CD4+ CD25+ T cells inhibit the activation of CD8+ responders by inhibiting both IL-2 production and up-regulation of IL-2Rα-chain (CD25) expression. Suppression is mediated via a TT interaction as activated CD4+ CD25+ T cells suppress the responses of TCR-transgenic CD8+ T cells stimulated with soluble peptide-MHC class I tetramers in the complete absence of APC. These results broaden the immunoregulatory role played by CD4+ CD25+ T cells in the prevention of autoimmune diseases, but also raise the possibility that they may hinder the induction of effector CD8+ T cells to tumor or foreign Ags.