The complete amino acid sequence of papain-solubilized HLA-B27, an antigen that presents a very strong association to the development of ankylosing spondylitis, has been determined. The overall sequence homology with the cross-reactive allelic products HLA-B7 and HLA-B40 (Bw60) is 93% and 92%, respectively. Half of the differences between HLA-B27 and -B7 are located in segments 63-83 and 113-116. Most of the known HLA class I antigens are different in these segments, and it is suggested that the corresponding residues may be involved in the alloantigenic determinants of HLA-B27. A free cysteine residue is present at position 67, and it is at least partially exposed to solvent. In addition, other differences are found in various areas of the two N-terminal domains. The comparison with available HLA class I sequences allows an evaluation of their contribution to the antigenic polymorphism of these molecules. The relevance of these data is discussed in connection with the mapping of functional sites of HLA class I antigens and with the association between HLA-B27 and ankylosing spondylitis.