Expression of HLA-DO (DO) in cells that express HLA-DM (DM) results in an altered repertoire of MHC class II/peptide complexes, indicating that DO modulates DM function. Human and murine B cells and thymic epithelial cells express DO, while monocytes/macrophages do not. Monocyte-derived dendritic cells (DC) also have been found to be DO-negative, leading to the assumption that DC do not express DO. In this study, we report that, in fact, certain types of human primary DC express DO. These include Langerhans cells (LC) and some subtypes of circulating blood DC. Specifically, the majority of BDCA-3(+) DC, a small subset of uncertain function, are DO(+), while smaller proportions of CD11c(+), BDCA-1(+) (myeloid) DC, at most a minority of CD123(+)/BDCA-2(+) (plasmacytoid) DC, and no detectable CD16(+) (myeloid) DC, express DO. Immunohistochemistry of human tonsil sections demonstrates that tonsillar interdigitating DC are also DO(+). In a subset of immature LC with higher DO expression, an increased fraction of surface DR molecules carry CLIP peptides, indicating that DO functions as a DM inhibitor in these cells. LC expression of DO is down-regulated by maturation stimuli. DM levels also decrease under these conditions, but the DM:DO ratio generally increases. In the myeloid cell types tested, DO expression correlates with levels of DObeta, but not DOalpha, implying that modulation of DObeta regulates DO dimer abundance in these cells. The range of APC types shown to express DO suggests a broader role for DO in immune function than previously appreciated.