Updated project metadata. Assessing the self-peptides presented by susceptible major histocompatibility complex (MHC) molecules is crucial for evaluating the pathogenesis and therapeutics of tissue-specific autoimmune diseases. However, direct examination of such MHC-bound peptides displayed in the target organ remains largely impractical. Here, we demonstrate that the blood leukocytes from non-obese diabetic (NOD) mice presented peptide epitopes to autoreactive CD4 T cells. These peptides were bound to the autoimmune class II MHC molecule, I-Ag7, and originated from insulin B-chain and C-peptide. The presentation required a glucose challenge, which stimulated the release of insulin peptides from pancreatic islets. The circulating leukocytes, especially the B cells, promptly captured and presented these peptides. Although canonical intracellular processing of insulin was involved in the presentation, extracellular binding of catabolized insulin products to I-Ag7 gave rise to a unique pathogenic epitope. Administration of monoclonal antibodies recognizing insulin B-chain abolished the presentation and diminished diabetes incidence. Mass spectrometry analysis of the leukocyte MHC-II peptidomes revealed a series of beta cell derived peptides, with identical sequences to those previously in the islet MHC-II peptidome. Thus, the WBC peptidome echoes that found in islets and serves to identify immunogenic peptides in an otherwise inaccessible tissue.