We have determined the crystal structure of I-A^g7, an integral component in murine type I diabetes development. Several features distinguish I-A^g7 from other non-autoimmune-associated MHC class II molecules, including novel peptide and heterodimer pairing interactions. The binding groove of I-A^g7 is unusual at both terminal ends, with a potentially solvent-exposed channel at the base of the P1 pocket and a widened entrance to the P9 pocket. Peptide binding studies with variants of the hen egg lysozyme I-A^g7 epitope HEL(11–25) support a comprehensive structure-based I-A^g7 binding motif. Residues critical for T cell recognition were investigated with a panel of HEL(11–25)-restricted clones, which uncovered P1 anchor–dependent structural variations. These results establish a framework for future experiments directed at understanding the role of I-A^g7 in autoimmunity.