A diabetes-associated peptide in the glutamic acid decarboxylase 65 (GAD65) molecule, p524-543, activates two distinct populations of T cells, which apparently play opposite roles in the development of diabetes in NOD mice. By comparing the fine specificity of these two T cell repertoires using a nested set of truncated peptides that cover the p524-543 region, we found, surprisingly, that all clones recognized the same core within this peptide, p530-539. The core itself was non-immunogenic, but the residues flanking this shared sequence played the crucial role in selecting T cells to activate. A peptide missing N-terminal flanking residues at position 528 and 529 was stimulatory in NOD but not in MHC-matched, NOD-resistant (NOR) mice, suggesting that a protective response in the resistant mice may require T cell recognition of one or more of the N-terminal flanking residues. T cell repertoire studies demonstrated selective clonal expansions within the BV4 TCR family that dominates the p524-543 response in NOD but not in NOR mice. These data suggest that processing or trimming events affecting T cell recognition of very few flanking residues of diabetes-associated determinants might be involved in the protective response in NOR mice.