Adaptive immune responses are tailored to different types of pathogens through differentiation of naive CD4 T cells into functionally distinct subsets of effector T cells (T helper 1 (T_H 1), T_H 2, and T_H 17) defined by expression of the key transcription factors T-bet, GATA3, and RORγt, respectively. Regulatory T (T_reg ) cells comprise a distinct anti-inflammatory lineage specified by the X-linked transcription factor Foxp3 (refs 2, 3). Paradoxically, some activated T_reg cells express the aforementioned effector CD4 T cell transcription factors, which have been suggested to provide T_reg cells with enhanced suppressive capacity. Whether expression of these factors in T_reg cells-as in effector T cells-is indicative of heterogeneity of functionally discrete and stable differentiation states, or conversely may be readily reversible, is unknown. Here we demonstrate that expression of the T_H 1-associated transcription factor T-bet in mouse T_reg cells, induced at steady state and following infection, gradually becomes highly stable even under non-permissive conditions. Loss of function or elimination of T-bet-expressing T_reg cells-but not of T-bet expression in T_reg cells-resulted in severe T_H 1 autoimmunity. Conversely, following depletion of T-bet^- T_reg cells, the remaining T-bet⁺ cells specifically inhibited T_H 1 and CD8 T cell activation consistent with their co-localization with T-bet⁺ effector T cells. These results suggest that T-bet⁺ T_reg cells have an essential immunosuppressive function and indicate that T_reg cell functional heterogeneity is a critical feature of immunological tolerance.