The biosynthesis of the HLA-DR antigens was studied in the B lymphoblastoid cell line BRI 8. Three chains, of molecular weights 33,000 (alpha), 31,000 (p31), and 26,000 (beta) were detected intracellularly in pulse-labeled cells by immunoprecipitation with anti-(HLA-DR) sera. The alpha and beta chains were inserted asymmetrically into the rough endoplasmic reticulum as transmembrane polypeptides with the majority of the polypeptide chains oriented in the lumen. At this stage, both chains carried "high mannose" oligosaccharide units which were processed to the complex form during subsequent intracellular transport to the cell surface. The Mr = 31,000 polypeptide was also glycosylated but was structurally distinct from the alpha chain and was probably oriented differently in the lipid bilayer, with a much greater proportion of its polypeptide chain exposed in the cytoplasm. It ws not, therefore, a precursor of the alpha chain. The mature HLA-DR antigens at the plasma membrane comprised polypeptides of Mr = 34,000 and 28,000. These chains corresponded to the processed alpha and beta chains. Although the Mr = 31,000 component was only detected intracellularly, it was not ruled out that some or all of it may have been processed and exposed on the cell surface with an apparent molecular weight indistinguishable from that of the alpha chain.