H2-M is a major histocompatibility complex (MHC) class II-like molecule that catalyzes peptide binding to MHC class II molecules. Recently, the H2-O heterodimer, encoded by H2-Oa and H2-Ob in the MHC class II region, has been shown to be physically associated with H2-M in B cells and to downregulate H2-M function. Examination of H2-O expression in freshly isolated mouse organs revealed that H2-Oa- and H2-Ob-specific transcripts are present in both lymphoid and nonlymphoid tissues. To evaluate the gene regulation and functional impact of H2-O on antigen presentation, we examined the effects on MHCII, invariant chain (Ii), H2-M, and H2-O gene expression of interleukin (IL)-4, IL-10, and interferon (IFN)-gamma in different antigen-presenting cells (APCs). In nonprofessional APCs, e.g., L929 fibroblasts, IFN-gamma-inducible expression of the MHC class II-specific transcription factor CIITA is associated with coordinate expression of MHCII, Ii, H2-M, and H2-Oa genes but without concomitant H2-Ob induction. In contrast, professional APCs, e.g., the macrophage cell line P388D1, exhibit constitutive H2-Oa and H2-Ob expression, which is not inducible by IFN-gamma in contrast to CIITA, MHCII, Ii, and H2-M expression. In B cells, CIITA, MHCII, Ii, and H2-M genes are differentially expressed relative to H2-Oa and H2-Ob genes upon stimulation with IL-4, IL-10, or IFN-gamma. A differential ratio of H2-M to H2-O may represent one mechanism by which professional and nonprofessional APCs bypass H2-O inhibitory activity.