The predominant class II, or Ia, antigen of the human major histocompatibility complex is HLA-DR. It consists of an alpha and a beta chain, the latter being responsible for the remarkable polymorphism of these Ia antigens. Studies with cloned genes had shown the existence of more than one DR beta-chain locus. We have isolated about 100 kilobases of the HLA-DR beta-chain gene region from a cosmid library generated from a consanguineous homozygous B-cell line of the DR3 haplotype. Three HLA-DR beta-chain genes have been characterized. They are arranged in a head-to-tail orientation. One of the genes lacks the region encoding the first domain of the DR beta chain. The two other genes are transcribed, as shown by RNA blot hybridization analysis. A striking restriction site homology has been found within the DR beta-chain gene cluster, suggesting a recent duplication event involving at least 25 kilobases of DNA. Moreover, the molecular map of DR beta chain genes cloned from B-cell lines of two other HLA-DR haplotypes shows extensive homology between alleles of a given DR beta-chain locus.