Adoptive transfer of ovalbumin (OVA)-specific T cells from the DO.11 TCR transgenic mouse on a Rag^−/− background into mice expressing OVA in pancreatic islet cells induces acute insulitis and diabetes only if endogenous lymphocytes, including regulatory T cells, are removed. When wild-type OVA-specific/Rag^−/− T cells, which are all CD25⁻, are transferred into islet antigen–expressing mice, peripheral immunization with OVA in adjuvant is needed to induce diabetes. In contrast, naive CTLA-4^−/−/Rag^−/− OVA-specific T cells (also CD25⁻) develop into Th1 effectors and induce disease upon recognition of the self-antigen alone. These results suggest that CTLA-4 functions to increase the activation threshold of autoreactive T cells, because in its absence self-antigen is sufficient to trigger autoimmunity without peripheral immunization. Further, CTLA-4 and regulatory T cells act cooperatively to maintain tolerance, indicating that the function of CTLA-4 is independent of regulatory cells, and deficiency of both is required to induce pathologic immune responses against the islet self-antigen.