The eye is an immunologically privileged organ whose Ags serve as targets for experimental autoimmune uveitis (EAU), a model for human uveitis. We used a hydrodynamic iv injection of naked DNA to express the uveitogenic retinal Ag interphotoreceptor retinoid-binding protein (IRBP) in the periphery, thus revoking its immune-privileged status. IRBP was expressed in the liver within hours of administration of as little as 10 μg of IRBP-DNA. Vaccinated mice were highly protected from EAU induced by immunization with IRBP for at least 10 wk after vaccination. Protection was partial in a reversal protocol. Mechanistic studies revealed specific hyporesponsiveness to IRBP without immune deviation, no evidence for apoptosis either by the Fas-or Bcl-2-regulated (mitochondrial) pathway and apparent lack of dependence on CD8+ cells, IL-10, or TGF-β. In contrast, depletion of CD25+ cells after vaccination and before challenge markedly abrogated protection. IRBP-specific CD4+ CD25 high T cells could be cultured from vaccinated mice and transferred protection to unvaccinated, EAU-challenged recipients. In vitro characterization of these cells revealed that they are Ag specific, anergic, express FoxP3, CTLA-4, and glucocorticoid-induced TNFR, and suppress by contact. Thus, expression of IRBP in the periphery by DNA vaccination results in tolerance that acts at least in part through induction of IRBP-specific, FoxP3+ CD4+ CD25+ regulatory T cells. DNA vaccination may offer a new approach to Ag-specific therapy of uveitis.