Type 1 diabetes is an autoimmune disease mediated by β cell-specific CD4+ and CD8+ T cells. Tracking β cell-specific T cells is one approach to monitor the diabetogenic response in at risk or diabetic individuals. Such analyses, however, are limited to PBL because T cells infiltrating the pancreatic islets are normally inaccessible. A key issue is whether peripheral β cell-specific T cells accurately reflect those cells infiltrating the target tissue. We investigated the properties of CD4+ T cells specific for a mimetic epitope recognized by the BDC2. 5 clonotypic TCR in NOD mice. Soluble IA g7-Ig (sIA g7-Ig) multimer complexes covalently linked to a mimetic BDC peptide (sIA g7-mBDC) were used to identify or isolate CD4+ T cells from PBL and the islets of NOD mice. A temporal increase in sIA g7-mBDC binding (g7-mBDC+) T cells corresponding with the progression of β cell autoimmunity was detected in both PBL and islets in NOD female mice. In contrast to T cells in PBL, however, the majority of islet g7-mBDC+ T cells exhibited a type 1 phenotype, and mediated diabetes upon transfer into NOD. scid recipients. TCR-β and CDR-β gene usage of single islet-infiltrating g7-mBDC+ CD4+ T cells from individual NOD mice showed a restricted repertoire dominated by one or two clones typically expressing TCR β-chain variable TRBV-15. In contrast, a distinct and diverse TCR repertoire was detected for PBL-derived g7-mBDC+ T cells. These results demonstrate that PBL and islet CD4+ T cells specific for a given β cell epitope can differ regarding pathogenicity and TCR repertoire.