HLA-DO/H2-O is a highly conserved, nonpolymorphic MHC class II-like molecule expressed in association with H2-M in thymic epithelial cells, B lymphocytes, and primary dendritic cells. The physiological function of DO remains unknown. The finding of cell maturation-dependent DO expression in B lymphocytes and dendritic cells suggests the possibility that H2-O functions to promote the presentation of exogenous Ag by attenuating presentation of endogenous self-peptides. In the current study, we report that H2-O(-/-) mice spontaneously develop high titers of IgG2a/c antinuclear Abs (ANAs) with specificity for dsDNA, ssDNA, and histones. Reconstitution of RAG1(-)(/)(-) mice with T and B cells from H2-O(-)(/)(-) or wild-type mice demonstrated that production of ANAs requires participation of CD4(+) T cells from H2-O(-)(/)(-) mice. Bone marrow chimeras demonstrated that loss of H2-O expression in thymic epithelial cells did not induce ANAs, and that lack of H2-O expression in bone marrow-derived cells was sufficient to induce the autoimmune phenotype. Despite production of high titers of autoantibodies, H2-O(-/-) mice exhibit a delayed generation of humoral immunity to model Ags (OVA and keyhole limpet hemocyanin), affecting all major T-dependent Ig classes, including IgG2a/c. Ag presentation experiments demonstrated that presentation of exogenous Ag by H2-O(-/-) APC was inefficient as compared with wild-type APC. Thus, H2-O promotes immunity toward exogenous Ags while inhibiting autoimmunity. We suggest that H2-O, through spatially or temporally inhibiting H2-M, may enhance presentation of exogenous Ag by limiting newly generated MHC class II molecules from forming stable complexes with endogenous self-peptides.