Nonobese diabetic (NOD) mice spontaneously develop insulitis and destruction of pancreatic islet β cells similar to type 1 diabetes mellitis in humans. Insulitis also occurs in the BDC2. 5 TCR transgenic line of NOD mice that express the rearranged TCR α-and β-chain genes of a diabetogenic NOD CD4 T cell clone. When activated with syngeneic islet cells in culture, BDC2. 5 T cells adoptively transfer disease to NOD recipients, but the identity of the islet cell Ag responsible for pathogenicity is not known. To characterize the autoantigen (s) involved, BDC2. 5 T cells were used to screen a combinatorial peptide library arranged in a positional scanning format. We identified more than 100 decapeptides that stimulate these T cells at nanomolar concentrations; they are then capable of transferring disease to NOD-scid mice. Surprisingly, some of the peptides include sequences similar (8 of 10 residues) to those found within the 528–539 fragment of glutamic acid decarboxylase 65. Although this 12-mer glutamic acid decarboxylase 65 fragment is only slightly stimulatory for BDC2. 5 T cells (EC 50> 100 μM), a larger 16-mer fragment, 526–541, shows activity in the low micromolar range (EC 50= 2.3 μM). Finally, T cells from prediabetic NOD mice respond spontaneously to these peptide analogs in culture; this finding validates them as being related to a critical autoantigen involved in the etiology of spontaneous diabetes and indicates that their further characterization is important for a better understanding of underlying disease mechanisms.