The ability of peptides to form stable complexes with MHC class II molecules expressed in the host determines their ability to recruit CD4 T cells during an immune response. In this study, we sought to define the features of the antigenic peptides that control their kinetic stability with I-A(d) because of the diversity of peptides that this molecule is known to present. Peptide dissociation assays indicated that each pocket of I-A(d) displays exquisite sensitivity to side chain structure, size, and charge. Most surprising were results related to the P1 pocket, which has been difficult to define by conventional competition assays. Our studies revealed a considerable degree of specificity in the P1 pocket but also an unexpected degree of structural flexibility. Amino acids with neutral side chains such as Met and the alternatively negatively charged Glu are both highly favored at P1. Interestingly, these two options at the P1 pocket in I-A(d) display dramatically different pH-dependent interactions with the class II molecule. These findings are discussed in the context of a structural model to explain these data and in light of the immunological implications of pH-dependent behavior of class II-peptide complexes in acidic endosomal compartments, where DM-catalyzed loading of class II molecules takes place, and at the neutral pH of the APC cell surface, where class II-peptide complexes promote activation of CD4 T cells.