The inhibitory immunoregulatory receptor CTLA-4 is critical in maintaining self-tolerance, but the mechanisms of its actions have remained controversial. Here we examined the antigen specificity of tissue-infiltrating CD4⁺ T cells in Ctla4^−/− mice. After adoptive transfer, T cells isolated from tissues of Ctla4^−/− mice showed T cell antigen receptor (TCR)-dependent accumulation in the tissues from which they were derived, which suggested reactivity to tissue-specific antigens. We identified the pancreas-specific enzyme PDIA2 as an autoantigen in Ctla4^−/− mice. CTLA-4 expressed either on PDIA2-specific effector cells or on regulatory T cells was sufficient to control tissue destruction mediated by PDIA2-specific T cells. Our results demonstrate that both cell-intrinsic and non–cell-autonomous actions of CTLA-4 operate to maintain T cell tolerance to a self antigen.