The autoantigen glutamic acid decarboxylase 65 (GAD 65) is believed to be an important target antigen in insulin-dependent diabetes mellitus (IDDM), since an age-related spontaneous breakdown in tolerance is observed, and cell-mediated and autoantibody immune responses have been reported in humans and NOD mice. We sought to identify immunogenic epitopes of GAD 65 which are presented to T cells by the type I diabetes susceptibility allele (Ag7), using overlapping 15-mer synthetic peptides spanning the entire sequence of this protein. Four epitopes (p206 - 220, p221 - 235, p286 - 300, p571 - 585) were identified by screening a panel of T-cell hybridomas generated from GAD 65-immunized NOD mice. These immunogenic epitopes are unrelated to the previously described T-cell epitopes of GAD 65 reported in NOD mice. Of the GAD 65 amino acid sequence, 206 - 220 and 221 - 235 are the two most dominant T-cell epitopes identified in this study. Sixty-three percent and 25% of GAD 65-responding T cell hybridomas react to p206 - 220 and p221 - 235, respectively. The remaining two peptides (p286 - 300, p571 - 585) are less dominant T-cell responses. The identification of the whole spectrum of GAD 65 Ag7 epitopes should further the investigation of the role of this autoantigen in the pathogenesis of IDDM.