Two-dimensional gel electrophoretic analyses of immunoprecipitates of the HLA-D region antigens revealed three beta (Mr 28 X 10(3) to 30 X 10(3) structurally polymorphic polypeptides. Analyses of the nonglycosylated beta polypeptides indicated that their polymorphism is most probably due to variations in amino acid structure. The polymorphism has been examined using lymphoblastoid cell lines that were derived from different geographical populations (North European and Italian) and that expressed various HLA-DR alloantigenic specificities. The results confirm that the positional variation of one beta polypeptide (namely beta-1) correlated with the HLA-DR allospecificity and was not influenced by the ethnic origin of the individual donating the cells. The positional variation of a second beta polypeptide (namely beta-2) was consistent with strong linkage dis-equilibrium between the corresponding genetic locus and the HLA-DR locus. Further, the association with a particular HLA-DR allospecificity was population-dependent; that is, different beta-2 polypeptides were associated with the same HLA-DR specificity in different geographical populations. In contrast, the position of the third beta polypeptide (namely, beta-3) which corresponds to the HLA-DC locus, did not necessarily vary with the HLA-DR specificity; that is, apparently the same beta-3 polypeptide was associated with different HLA-DR specificities provided the cells were of the same ethnic origin. The data indicate that lymphoblastoid cells express at least three sets of HLA-D region antigens that are encoded by different loci including HLA-DR and DC.