B cells from leukemic rabbits preferentially use a single V(H) gene, V(H)1, in their VDJ gene rearrangements. To determine whether Ig-secreting B cells from normal rabbits also preferentially use V(H)1, we generated rabbit x mouse heterohybridomas that stably secreted rabbit Ig that expressed V(H)a allotypic specificities and analyzed the V(H) genes used in their VDJ gene rearrangements. We cloned the VDJ genes from nine heterohybridomas, and by comparing the restriction map of the DNA immediately 5' of the translational start site of these VDJ genes to that of the same region 5' of V(H)1, we showed that eight of the nine heterohybridoma clones use V(H)1. Comparison of the nucleotide sequences of the eight V(H)1-using VDJ genes with the nucleotide sequence of germ-line V(H)1 showed that each of them had somatically diversified. The diversified regions included clustered nucleotide changes and codon insertions and deletions, such as would be expected if the diversification process involved somatic gene conversion. We searched our database of germ-line V(H) genes for genes that could serve as donors for the gene conversion events, and we identified potential V(H) donor genes for five regions of diversification. One of these regions of diversification spanned at least 132 bp and included a codon insertion as well as 15 nucleotide changes. The data confirm that Ab diversity may be generated by a somatic gene conversion-like mechanism. The results directly demonstrate that Ig-secreting B cells from normal rabbits preferentially use V(H)1 in their VDJ gene rearrangements.