B lymphocytes express the nonclassical class II molecule HLA-DO, which modulates the peptide loading activity of HLA-DM in the endocytic pathway. Binding to HLA-DM is required for HLA-DO to egress from the endoplasmic reticulum (ER). To gain insights into the mode of action of DO and on the role of DM in ER release, we sought to identify DM-binding residues on DO. Our results show that DOalpha encompasses the binding site for HLA-DM. More specifically, mutation of residue DOalpha41 on an exposed lateral loop of the alpha1 domain affects the binding to DM, ER egress, and activity of DO. Using a series of chimeric DR/DO molecules, we confirmed the role of the alpha chain and established that a second DM-binding region is located C-terminal to the DOalpha80 residue, most probably in the alpha2 domain. Interestingly, after mutation of a buried proline (alpha11) on the floor of the putative peptide-binding groove, HLA-DO remained functional but became independent of HLA-DM for ER egress and intracellular trafficking. Collectively, these results suggest that the binding of HLA-DM to DOalpha allows the complex to egress from the ER by stabilizing intramolecular contacts between the N-terminal antiparallel beta-strands of the DOalphabeta heterodimer.