Extrathymically derived regulatory T cells (iTregs) protect against autoimmunity to tissue-specific Ags. However, whether Agspecific iTreg generation and function is limited to secondary lymphoid tissue or whether it can occur within the tissue-specific local environment of the cognate Ag remains unresolved. Mice expressing b-galactosidase (bgal) on a retina-specific promoter (bgal mice) in conjunction with mice expressing GFP and diphtheria toxin (DTx) receptor (DTR) under control of the Foxp3 promoter, and bgal-specific TCR transgenic (BG2) mice were used to examine this question. Local depletion (ocular DTx), but not systemic depletion (i.p. DTx), of bgal-specific iTregs enhanced experimental autoimmune uveoretinitis induced by activated bgalspecific effector T cells. Injections of small amounts of bgal into the anterior chamber of the eye produced similar numbers of bgal-specific iTregs in the retina whether the mouse was depleted of pre-existing, circulating Tregs. Taken together, these results suggest that protection from tissue-specific autoimmunity depends on the function of local Ag-specific iTregs and that the retina is capable of local, "on-demand" iTreg generation that is independent of circulating Tregs.