We describe here a pathway of presentation involving peptides or denatured proteins that generate unique peptide–MHC complexes. Such complexes select for non-conventional CD4 T cells. We have examined this pathway and the corresponding CD4 T cells in diabetic autoimmunity. Autoimmunity requires both the escape of self-reactive T cells from thymic selection and, importantly, suitable conditions in peripheral tissues that allow for activation of T cells. In the autoimmune diabetes of NOD mice, insulin reactive T cells are highly focused on a peptide, encompassing the 9–23 segment of the B chain (B:9–23) bound to I-Ag7. Examination of the B:9–23 reactive T cell repertoire revealed the presence of two independent sets of T cells that recognize this epitope. One set, called type A, reacted like conventional CD4 T cells, recognizing both processed insulin protein and soluble B:9–23 peptide presented by APC. These T cells were highly deleted in the thymus and poorly represented in the periphery. The second set, called type B, did not recognize processed insulin protein presented by APC, but reacted strongly to the presentation of soluble B:9–23 peptide. Notably, this set was not deleted in the thymus, abundant in the periphery and caused diabetes. Free insulin peptides generated a unique peptide–MHC complex not found after insulin processing. These two T cell subsets discriminated between two independent, overlapping registers found within the B:9–23 peptide. In the islets of Langerhans, beta cells constitutively generated proteolyzed peptides from insulin, which were taken up by intra-islet APC and presented to peptide-specific type B T cells. Thus, self-reactive T cells that escape thymic selection can become pathogenic in the target organ where high concentrations of antigen and/or differences in intracellular processing lead to the presentation of peptides bound in distinct registers from those found in the thymus.