CD4+ T cell responses to glutamic acid decarboxylase (GAD65) spontaneously arise in nonobese diabetic (NOD) mice before the onset of insulin-dependent diabetes mellitus (IDDM) and may be critical to the pathogenic process. However, since both CD4+ and CD8+ T cells are involved in autoimmune diabetes, we sought to determine whether GAD65-specific CD8+ T cells were also present in prediabetic NOD mice and contribute to IDDM. To refine the analysis, putative K d-binding determinants that were proximal to previously described dominant Th determinants (206–220 and 524–543) were examined for their ability to elicit cytolytic activity in young NOD mice. Naive NOD spleen cells stimulated with GAD65 peptides 206–214 (p206) and 546–554 (p546) produced IFN-γ and showed Ag-specific CTL responses against targets pulsed with homologous peptide. Conversely, several GAD peptides distal to the Th determinants, and control K d-binding peptides did not induce similar responses. Spontaneous CTL responses to p206 and p546 were mediated by CD8+ T cells that are capable of lysing GAD65-expressing target cells, and p546-specific T cells transferred insulitis to NOD. scid mice. Young NOD mice pretreated with p206 and p546 showed reduced CTL responses to homologous peptides and a delay in the onset of IDDM. Thus, MHC class I-restricted responses to GAD65 may provide an inflammatory focus for the generation of islet-specific pathogenesis and β cell destruction. This report reveals a potential therapeutic role for MHC class I-restricted peptides in treating autoimmune disease and revisits the notion that the CD4-and CD8-inducing determinants on some molecules may benefit from a proximal relationship.