HLA-A and HLA-B antigens are integral membrane glycoproteins which consist of a glycosylated heavy chain embedded in the membrane in noncovalent association with beta 2-microglobulin, a water-soluble polypeptide. The assembly and maturation of these antigens has been studied in vivo in the human B lymphoblastoid cell line T5-1 (HLA-A1, -A2, -B8, -B27). Two antigenically distinct populations of HLA-A and -B heavy chains can be detected by antisera which recognize determinants sensitive to the conformation of the heavy chain. One heavy chain population is associated with beta 2-microglobulin, whereas the other population is not. These populations can be further distinguished by their oligosaccharide structure and their localization within the cell. Pulse-chase experiments demonstrate a precursor-product relationship between these heavy chain populations and suggest the following pathway for the assembly and maturation of HLA-A and -B antigens. The completed heavy chains initially carry high mannose oligosaccharides and are largely or wholly associated with beta 2-microglobulin. During the next 10-15 min, association with beta 2-microglobulin occurs and the heavy chain conformation is altered. Beginning at about 30 min after synthesis, the oligosaccharides are converted from the high mannose form to the complex form, and between 60 and 80 min after synthesis, the mature antigens appear at the cell surface. These observations are discussed in relation to in vivo and in vitro studies on the biosynthesis of a variety of secreted proteins and membrane proteins.