Peptide loading of major histocompatibility class II molecules is catalyzed in late endosomal and lysosomal compartments of cells by the catalytic action of human leukocyte antigen (HLA)‐DM (H‐2M in mice). In B cells, dendritic cells and thymic epithelial cells, the peptide loading of class II molecules is modified by the expression of the non‐classical class II molecule, HLA‐DO (H‐2O in mice). Collectively, studies to date support that DO/H‐2O expression inhibits the presentation of antigens acquired by cells via fluid phase endocytosis. However, in B cells, the expression of H‐2O promotes the presentation of antigens internalized by the B‐cell receptor. In this review, we summarize the literature pertaining to DO assembly, transport, and function, with an emphasis on the function of DO/H‐2O.