We demonstrate in this study the great degree of specificity in peptides selected by a class II MHC molecule during processing. In this specific case of the diabetogenic IA g7 molecule, the P9 pocket of IA g7 plays a critical role in determining the final outcome of epitope selection, a conclusion that is important in interpreting the role of this molecule in autoimmunity. Specifically, we examined the display of naturally processed peptides from APCs expressing either IA g7 molecules or a mutant IA g7 molecule in which the β57Ser residue was changed to an Asp residue. Using mass spectrometry analysis, we identified over 50 naturally processed peptides selected by IA g7-expressing APCs. Many peptides were selected as families with a core sequence and variable flanks. Peptides selected by IA g7 were unusually rich in the presence of acidic residues toward their C termini. Many peptides contained short sequences of two to three acidic residues. In binding analysis, we determined the core sequences of many peptides and the interaction of the acidic residues with the P9 pocket. However, different sets of peptides were isolated from APCs bearing a modified IA g7 molecule. These peptides did not favor acidic residues toward the carboxyl terminus.