Although the association between human histocompatibility leukocyte antigen (HLA) B27 and ankylosing spondylitis is the prototype of HLA-disease association, the mechanism underlying these associations has not been determined. We have investigated the possibility that the B27 molecules from patients with ankylosing spondylitis are different from those of normals, and only the “different” molecules predispose the individual to disease. Biosynthetically radiolabeled HLA-B27 molecules from patients with ankylosing spondylitis and normal individuals were compared by two-dimensional gel electrophoresis and tryptic peptide mapping with high pressure liquid chromatography. Extensive charge heterogeneity in the 45,000-dalton heavy chain was detected when B27 molecules were analyzed by two-dimensional gel electrophoresis; the charge heterogeneity was reduced, but not eliminated, when the B27 molecules were treated with neuraminidase to remove sialic acid residues before analysis. No structural difference in the B27 molecules from an ankylosing spondylitis patient and a normal individual were detected by two-dimensional gel electrophoresis. Analysis of [3H]leucine-labeled and [3H]arginine-labeled tryptic peptides and chymotryptic peptides of the trypsin insoluble material by reverse-phase high pressure liquid chromatography revealed identity of the B27 molecules from ankylosing spondylitis patients and normal individuals. These studies indicate that development of akylosing spondylitis in only some B27 positive individuals is not attributable to those individuals possessing variant B27 molecules.