The exchange of HLA class II-associated invariant chain peptides (CLIP) for cognate peptide is catalyzed by HLA-DM under acidic conditions in vitro by an unknown mechanism. Here, we show an association between HLA-DM and HLA-DR in vivo by coprecipitation of the two heterodimers. The association is favored at low pH and in the nonionic detergent digitonin. Most DM-DR complexes are isolated from dense subcellular fractions. Recovery of HLA-DM by the conformation-dependent DR3 monoclonal antibody 16.23 suggests an association with HLA-DR heterodimers beyond the stage at which CLIP is released. The additional N-linked glycan on mutant DR3 molecules isolated from the 10.24.6 cell line, which interferes with DM-enhanced CLIP release from DR3 in vitro, also affects the DM-DR interaction.