OBJECTIVE-Weak major histocompatibility complex (MHC) binding of self-peptides has been proposed as a mechanism that may contribute to autoimmunity by allowing for escape of autoreactive T-cells from the thymus. We examined the relationship between the MHC-binding characteristics of a β-cell antigen epitope and T-cell autoreactivity in a model of autoimmune diabetes. RESEARCH DESIGN AND METHODS-The binding of a proinsulin epitope, proinsulin-1(47-64) (PI-1[47-64]), to the MHC class II molecules I-A g7 and I-A k was measured using purified class II molecules. T-cell reactivity to the proinsulin epitope was examined in I-A g7+ and I-A k+ mice. RESULTS-C-peptide epitopes bound very weakly to I-A g7 molecules. However, C-peptide-reactive T-cells were induced after immunization in I-A g7-bearing mice (NOD and B6.g7) but not in I-A k-bearing mice (B10.BR and NOD.fo4). T-cells reactive with the PI-1(47-64) peptide were found spontaneously in the peripancreatic lymph nodes of pre-diabetic NOD mice. These T-cells were activated by freshly isolated β-cell in the presence of antigen-presenting cells and caused diabetes when transferred into NOD.scid mice. CONCLUSIONS-These data demonstrate an inverse relationship between self-peptide-MHC binding and T-cell autoreactivity for the PI-1(47-64) epitope in autoimmune diabetes.