In humans, the kappa light chain variable region gene (Vk) locus evolved in part by duplications of four large segments. Similarly, some portions of the human heavy chain constant region gene locus are duplicated. Recently, we found that the Humhv3005 Vh gene is highly homologous to the reported 1.9III gene. Subsequently, restriction fragment length polymorphism study of the human Vh locus with a 1.6-kb EcoR1 fragment downstream of the hv3005 gene (termed hv3005/E1.6) suggested that the hv3005 and the 1.9III Vh loci might be generated by duplication from a common-ancestor Vh gene segment. To assess this possibility, we mapped the 15-kb region of the isolated hv3005 clone, beginning from 2 kb upstream, and sequenced the adjacent Vh4 gene (designated Humhv4005) located 10 kb downstream of the hv3005 gene. The result showed that hv4005 shared 99% homology with the 1.9II gene, located about 11 kb downstream of the 1.9III gene. Taken together, these data demonstrate that the hv3005-hv4005 region and the 1.9III-1.9II region arose by a duplication of a common ancestor Vh gene segment.