H2-O is a nonpolymorphic class II molecule whose biological role remains to be determined. H2-O modulates H2-M function, and it has been generally believed to be expressed only in B lymphocytes and thymic medullary epithelial cells, but not in dendritic cells (DCs). In this study, we report identification of H2-O expression in primary murine DCs. Similar to B cells, H2-O is associated with H2-M in DCs, and its expression is differentially regulated in DC subsets as well as during cell maturation and activation. Primary bone marrow DCs and plasmacytoid DCs in the spleen and lymph nodes express MHC class II and H2-M, but not the inhibitor H2-O. In contrast, myeloid DCs in secondary lymphoid organs express both H2-M and H2-O. In CD8αα+ DCs, the ratio of H2-O to H2-M is higher than in CD8αα− DCs. In DCs generated from GM-CSF-and IL-4-conditioned bone marrow cultures, H2-O expression is not detected regardless of the maturation status of the cells. Administration of LPS induces in vivo activation of myeloid DCs, and this activation is associated with down-regulation of H2-O expression. Primary splenic DCs from H2-O−/− and H2-O+/+ mice present exogenous protein Ags to T cell hybridomas similarly well, but H2-O−/− DCs induce stronger allogeneic CD4 T cell response than the H2-O+/+ DCs in mixed leukocyte reactions. Our results suggest that H2-O has a broader role than previously appreciated in regulating Ag presentation.