OBJECTIVE: It is well established that the primary mediators of beta-cell destruction in type 1 diabetes are T-cells. Nevertheless, the molecular basis for recognition of beta-cell-specific epitopes by pathogenic T-cells remains ill defined; we seek to further explore this issue. RESEARCH DESIGN AND METHODS: To determine the properties of beta-cell-specific T-cell receptors (TCRs), we characterized the fine specificity, functional and relative binding avidity/affinity, and diabetogenicity of a panel of GAD65-specific CD4(+) T-cell clones established from unimmunized 4- and 14-week-old NOD female mice. RESULTS: The majority of GAD65-specific CD4(+) T-cells isolated from 4- and 14-week-old NOD female mice were specific for peptides spanning amino acids 217-236 (p217) and 290-309 (p290). Surprisingly, 31% of the T-cell clones prepared from 14-week-old but not younger NOD mice were stimulated with both p217 and p290. These promiscuous T-cell clones recognized the two epitopes when naturally processed and presented, and this dual specificity was mediated by a single TCR. Furthermore, promiscuous T-cell clones demonstrated increased functional avidity and relative TCR binding affinity, which correlated with enhanced islet infiltration on adoptive transfer compared with that of monospecific T-cell clones. CONCLUSIONS: These results indicate that promiscuous recognition contributes to the development of GAD65-specific CD4(+) T-cell clones in NOD mice. Furthermore, these findings suggest that T-cell promiscuity reflects a novel form of T-cell avidity maturation.