OBJECTIVE—CD8+ T-cells specific for islet antigens are essential for the development of type 1 diabetes in the NOD mouse model of the disease. Such T-cells can also be detected in the blood of type 1 diabetic patients, suggesting their importance in the pathogenesis of the human disease as well. The development of peptide-based therapeutic reagents that target islet-reactive CD8+ T-cells will require the identification of disease-relevant epitopes.RESEARCH DESIGN AND METHODS—We used islet-infiltrating CD8+ T-cells from HLA-A*0201 transgenic NOD mice in an interferon-γ enzyme-linked immunospot assay to identify autoantigenic peptides targeted during the spontaneous development of disease. We concentrated on insulin (Ins), which is a key target of the autoimmune response in NOD mice and patients alike.RESULTS—We found that HLA-A*0201-restricted T-cells isolated from the islets of the transgenic mice were specific for Ins1 L3–11, Ins1 B5–14, and Ins1/2 A2–10. Insulin-reactive T-cells were present in the islets of mice as young as 5 weeks of age, suggesting an important function for these specificities early in the pathogenic process. Although there was individual variation in peptide reactivity, Ins1 B5–14 and Ins1/2 A2–10 were the immunodominant epitopes. Notably, in vivo cytotoxicity to cells bearing these peptides was observed, further confirming them as important targets of the pathogenic process.CONCLUSIONS—The human versions of B5–14 and A2–10, differing from the murine peptides by only a single residue, represent excellent candidates to explore as CD8+ T-cell targets in HLA-A*0201–positive type 1 diabetic patients.