DM actions as a class II chaperone promote capture of diverse peptides inside the endocytic compartment (s). DM mutant cells studied to date express class II bound by class II-associated invariant chain-derived peptide (CLIP), a short proteolytic fragment of the invariant chain, and exhibit defective peptide-loading abilities. To evaluate DM functional contributions in k haplotype mice, we engineered a novel mutation at the DMa locus via embryonic stem cell technology. The present experiments demonstrate short-lived A k/CLIP complexes, decreased A k surface expression, and enhanced A k peptide binding activities. Thus, we conclude that DM loss in k haplotype mice creates a substantial pool of empty or loosely occupied A k conformers. On the other hand, the mutation hardly affects E k activities. The appearance of mature compact E k dimers, near normal surface expression, and efficient Ag presentation capabilities strengthen the evidence for isotype-specific DM requirements. In contrast to DM mutants described previously, partial occupancy by wild-type ligands is sufficient to eliminate antiself reactivity. Mass spectrometry profiles reveal A k/CLIP and a heterogeneous collection of relatively short peptides bound to E k molecules. These experiments demonstrate that DM has distinct roles depending on its specific class II partners.