The biosynthesis of the HLA-A and -B antigens was studied in the B lymphoblastoid cell line BRI 8 and the Burkitt lymphoma line DAudi. The heavy chains of the HLA-A and -B antigens were inserted asymmetrically into the rough endoplasmic reticulum of BRI 8 cells as transmembrane polypeptides. During the first 5 min after synthesis, the majority of the heavy chains became associated with beta 2-microglobulin. At this stage, the heavy chains carried a high mannose (core) oligosaccharide. Subsequent processing of the oligosaccharide unit occurred during the next 20 to 25 min, resulting in the conversion of the oligosaccharide from the high mannose to the complex form. About 30 to 40 min after synthesis, the mature antigens were expressed at the cell surface. Glycosylation was not required for asymmetric insertion, intracellular transport, or surface expression of the HLA-A and -B antigens. However, studies with Daudi cells indicated that combination with beta 2-microglobulin was necessary for subsequent processing and intracellular transport of the heavy chains after their synthesis in the rough endoplasmic reticulum.