In Heterodontus, a phylogenetically primitive shark species, the variable (VH), diversity (DH), joining (JH) segments, and constant (CH) exons are organized in individual approximately 18-20-kb "clusters." A single large VH family with > 90% nucleic acid homology and a monotypic second gene family are identified by extensive screening of a genomic DNA library. Little variation in the nucleotide sequences of DH segments from different germline gene clusters is evident, suggesting that the early role for DH was in promoting junctional diversity rather than contributing unique coding specificities. A gene-specific oligodeoxynucleotide screening method was used to relate specific transcription products (cDNAs) to individual gene clusters and showed that gene rearrangements are intra- rather than intercluster. This provides further evidence for restricted diversity in the immunoglobulin heavy chain of Heterodontus, from which it is inferred that combinatorial diversity is a more recently acquired means for generating diversity. The observed differences between cDNA sequences selected and the sequences of segmental elements derived from conventional genomic libraries as well as from VH segment-specific libraries generated by direct PCR amplification of genomic DNA indicate that the VH repertoire is diversified by both junctional diversity and somatic mutation. Taken together, these findings suggest a heretofore unrecognized contribution of somatic variation that preceded both extensive diversification of the germline repertoire and the combinatorial joining process in the evolution of humoral immunity.