HLA-DO is an intracellular nonclassical MHC class II molecule expressed in the endocytic pathway of B lymphocytes. It shapes the repertoire of peptides bound to classical class II molecules such as HLA-DR by regulating the activity of HLA-DM. Using a peptide corresponding to the cytoplasmic tail of HLA-DO(beta), we have developed a mouse monoclonal antibody, HKC5. Immunofluorescence microscopy revealed that HKC5 recognizes HLA-DO molecules present in the endoplasmic reticulum as well as those in vesicular compartments of the endocytic pathway. In addition, the antibody detects the isolated beta chain on Western blots. Using mutants of the DO(beta) cytoplasmic tail fused to a reporter molecule and expressed in epithelial cells, we showed by flow cytometry that the antibody epitope includes one or both of the leucine residues forming the lysosomal sorting signal. Finally, we have used HKC5 to evaluate the presence of the HLA-DO(beta) chain in HeLa cells expressing the class II transactivator protein CIITA. Our flow cytometry and confocal microscopy analyses showed a marked expression of DO(beta) suggesting that HLA-DO could accumulate under the influence of CIITA in non-B cells.