The UGT1 locus is felt to be highly conserved between species, as is evident from the characterization of the locus in rodents and humans. In rabbits, cDNAs encoding proteins homologous to human UGT1A4, UGT1A6, and UGT1A7 have previously been identified. Here we demonstrate by Southern blot analysis, using exon 1 divergent 5' segments from rabbit UGT1A4 and UGT1A6 cDNAs, the existence of a cluster of highly related genes that are homologous to each of these exon 1 sequences. In comparing rabbit and human, it is evident that the UGT1A4 and UGT1A6 gene clusters in rabbit have undergone gene duplication. This is particularly evident with rabbit UGT1A6. The human UGT1A6 cDNA anneals to only a single gene fragment, as displayed by Southern blot analysis, indicating that the UGT1A6 exon 1 sequence is highly conserved. However, up to six rabbit UGT1A6 genes could be predicted from Southern blot analysis. To examine the potential linkage of the rabbit UGT1A6 genes, multiple UGT1A6 exons were identified from genomic DNA by extended polymerase chain reaction techniques and cloning of the UGT1A6 exon 1 sequences. Five unique UGT1A6 exon 1 gene sequences were characterized that could be predicted to encode proteins that are 98% similar in amino acid structure. Using a conserved region of the rabbit UGT1A6 cDNA as a probe to screen cDNA libraries, we identified a second UGT1A6 cDNA, termed UGT1A6alpha. In addition, a cDNA that encodes a protein similar to human UGT1A3 was also cloned. Characterization of UGT1A6alpha demonstrated the protein to be 98.9% identical to UGT1A6. The expression of rabbit UGT1A3, UGT1A4, and UGT1A6 displayed catalytic activities similar to their human orthologs. However, UGT1A6alpha was catalytically divergent from UGT1A6, indicating that UGT1A6 and UGT1A6alpha do not arise from allelic polymorphism. These results demonstrate that lagomorphs have evolved at least five additional UGT1A6 genes, an event that is not duplicated in rodents or humans.