Class II presentation mutants have mutations in the HLA-DMA or B genes and are defective in the presentation of whole exogenous Ags restricted by HLA-DR, -DQ, and -DP. The functional defect in Ag presentation is accompanied by an altered conformation of cell surface class II molecules and instability of extracted class II dimers in SDS-PAGE; the latter can be corrected by incubation of mutant cells in an acidic pH in the presence of cognate peptide. Here we investigated the basis for correction of class II dimer instability by acid/cognate peptide treatment and the extent to which this treatment corrects the class II conformational defect in DMB mutants. We found that an acidic pH generates peptide binding sites in class II molecules of DMB mutants by eluting invariant chain (li)-derived peptides from them. Cognate peptides can then bind to the empty binding sites of class II molecules in a pH-independent manner, which results in stabilization of class II dimers. Acid/peptide treatment also restores the DR polymorphic epitope recognized by mAb 220.127.116.11 but not the DR polymorphic epitope recognized by mAb 16.23; low pH gradually destroys the 16.23 epitope in nonmutant cells. Mutant 10.24.6, which has a mutation in the DRA coding region creating an extra glycosylation site, also has unstable DR dimers whose stability is restored by acid/peptide treatment. These results suggest that the primary phenotypic defect in both the DMB and 10.24.6 mutants is the abundance of li peptides and lack of cognate peptides bound to class II molecules.