The human UDP-glucuronosyltransferases, UGT1A8, 1A9, and 1A10, are closely related in sequence and have a major role in the elimination of lipophilic chemicals by glucuronidation. UGT1A8 and 1A10 are expressed exclusively in the gastrointestinal tract, whereas UGT1A9 is expressed mainly in the liver and kidneys. To determine the factors contributing to the extrahepatic expression of these UDP-glucuronosyltransferases, we have cloned and characterized the promoters of the UGT1A8, 1A9, and 1A10 genes and studied their regulation in the colon cell line, Caco2. Their transcription start sites were mapped, and a functional overlapping Sp1/initiator-like site was identified which strongly contributed to UGT1A8 and 1A10 promoter activity. The high promoter activity of UGT1A8 and 1A10 correlated with the binding of nuclear proteins (complex B) to this region. Two-bp differences in the corresponding site in the UGT1A9 promoter prevented the binding of complex B and reduced promoter activity. Although Sp1 was able to bind to the Sp1/initiator-like site, its binding was dispensable for promoter activity. However, the binding of Sp1 to a second Sp1 site 30 bp 5' to the Sp1/initiator-like site greatly enhanced the activity of the UGT1A8 and 1A10 promoters. These results provide evidence that the UGT1A8, 1A9, and 1A10 genes are differentially regulated through an initiator element in their 5'-flanking regions.