Pemphigus vulgaris is a severe autoimmune disease caused by autoantibodies against the cutaneous adhesion molecule, desmoglein 3 (Dsg3). The aim of this study was to characterize the specificity of autoreactive Th cells, which presumably regulate Dsg3-specific autoantibody production. Ninety-seven Th1 and Th2 clones isolated from 16 pemphigus patients and 12 HLA-matched healthy donors recognized the Dsg3 peptides, DG3 (78-94), DG3 (96-112), DG3 (189-205), DG3 (205-221), and DG3 (250-266). Peptide DG3 (96-112), and to a lesser extent DG3 (250-266), was recognized by the majority of T cells from patients and healthy donors in association with HLA-DRB1* 0402 and DQB1* 0503 which were prevalent in the pemphigus patients and Dsg3-responsive healthy donors. Analyzing the Vβ-chain of the TCR of the DG3 (96-112)-specific T cells showed no restricted TCR usage. Peptides DG3 (342-358) and DG3 (376-392) were exclusively recognized by T cell clones (n= 13) from patients while DG3 (483-499) was only recognized by T cell clones (n= 3) from a healthy donor. All Dsg3 peptides contained conserved amino acids at relative positions 1, 4, and 6; amino acids with a positive charge at position 4 presumably represent anchor motifs for DRB1* 0402. These findings demonstrate that T cell recognition of distinct Dsg3 peptides is restricted by distinct HLA class II molecules and is independent from the development of pemphigus vulgaris.