CD4+ CD25+ T cells represent a unique population of “professional” suppressor T cells that prevent induction of organ-specific autoimmune disease. In vitro, CD4+ CD25+ cells were anergic to simulation via the TCR and when cultured with CD4+ CD25− cells, markedly suppressed polyclonal T cell proliferation by specifically inhibiting the production of IL-2. Suppression was cytokine independent, cell contact dependent, and required activation of the suppressors via their TCR. Further characterization of the CD4+ CD25+ population demonstrated that they do not contain memory or activated T cells and that they act through an APC-independent mechanism. CD4+ CD25+ T cells isolated from TCR transgenic (Tg) mice inhibited responses of CD4+ CD25− Tg T cells to the same Ag, but also inhibited the Ag-specific responses of Tg cells specific for a distinct Ag. Suppression required that both peptide/MHC complexes be present in the same culture, but the Ags could be presented by two distinct populations of APC. When CD4+ CD25+ T cells were cultured with anti-CD3 and IL-2, they expanded, remained anergic, and in the absence of restimulation via their TCR, suppressed Ag-specific responses of CD4+ CD25− T cells from multiple TCR transgenics. Collectively, these data demonstrate that CD4+ CD25+ T cells require activation via their TCR to become suppressive, but once activated, their suppressor effector function is completely nonspecific. The cell surface molecules involved in this TT interaction remain to be characterized.