Pemphigus vulgaris (PV) is a severe autoimmune bullous skin disorder and is primarily associated with circulating autoantibodies against desmoglein 3 (Dsg3) that are presumably regulated by Th cells. The aim of this study was to identify Dsg3-specific T regulatory (Tr) cells that may help to maintain and restore natural tolerance against Dsg3. Dsg3-responsive IL-10-secreting Tr1 cells were isolated by MACS cytokine secretion assay from healthy carriers of the PV-associated HLA class II alleles, DRB1* 0402 and DQB1* 0503, but were only rarely detected in PV patients. The Dsg3-specific Tr1 cells secreted IL-10, TGF-β, and IL-5 upon Ag stimulation, proliferated in response to IL-2 but not to Dsg3 or mitogenic stimuli, and inhibited the proliferative response of Dsg3-and tetanus toxoid-responsive Th clones in an Ag-specific (Dsg3) and cell number-dependent manner. Moreover, their inhibitory effect was blocked by Ab against IL-10, TGF-β, and by paraformaldehyde fixation. These observations strongly suggest that 1) Dsg3-responsive Tr1 cells predominate in healthy individuals, 2) their growth requires the presence of IL-2, and 3) they exert their Dsg3-dependent inhibitory function by the secretion of IL-10 and TGF-β. Because autoaggressive T cells responsive to identical epitopes of Dsg3 were recently found both in PV patients and healthy individuals, the identified Tr1 cells may be critically involved in the maintenance and restoration of tolerance against Dsg3.