To understand the relative importance of germ-line genes in the generation of the functional human antibody repertoire, it is first necessary to define the number of variable region genes and to determine their fine structure. We have focused on the human VkIII variable region gene family because of its association with autoantibodies. A human genomic library was screened with a VkIII cDNA probe and subsequently with a VkIII germ-line gene probe. Seven different VkIII clones were isolated and characterized by restriction mapping and sequence analyses. Three clones have identical restriction enzyme sites over a 12-kilobase (kb) region, contain identical sequences over an 895-base pair (bp) region, and thus are likely to be different isolates of the same human VkIII gene. Another two clones have identical restriction enzyme sites over a 5-kb region, are identical over a stretch of 905 bp sequenced, and likely represent independent isolates of another human VkIII gene. The remaining two VkIII clones consist of two additional VkIII genes which are homologous to each other, but are quite different from the first two VkIII genes. Thus, four new human VkIII genes were defined. Together with four other VkIII genes previously isolated by other investigators, a total of eight human VkIII germ-like genes have now been described. A comparison of the deduced amino acid sequences of these genes with the reported amino acid sequences of all human VkIII light chains suggests that at least one additional VkIII gene exists in the germ line. Among the eight identified human germ-line VkIII genes, three are pseudogenes. Of the remaining five potential functional genes, one gene seems to encode a majority of the VkIII light chains which have been sequenced. Possible explanations for this phenomenon are discussed.