The diabetes-susceptible class II MHC genes (in human and mouse) share unique nonaspartic acid residues at position 57 of the class II β-chain. Transgenic expression of a mutant IA g7, substituting histidine and serine at position 56 and 57 of β-chain with proline and aspartic acid (IA g7 PD), respectively, inhibits diabetes development in the nonobese diabetic mouse model. Here, we demonstrate that immature thymocytes expressing a diabetogenic islet Ag-specific transgenic TCR are positively selected by IA g7 PD class II MHC to give rise to mature CD4+ T cells. However, splenic APCs expressing the same IA g7 PD fail to present pancreatic islet Ag to mature T cells bearing this diabetogenic TCR. These results indicate that nonaspartic acid residues at position 57 of class II MHC β-chain is important for diabetogenic CD4+ T cell activation in the periphery but is not essential for the formation of a diabetogenic T cell repertoire in the thymus.