The human UDP-glucuronosyltransferases (UGT) -1A8 and -1A10 are exclusively expressed in extrahepatic tissues and primarily in the gastrointestinal tract, whereas UGT1A9 is expressed mainly in the liver and kidneys. We have demonstrated previously that the UGT1A8 and UGT1A10 genes, in contrast to the UGT1A9 gene, are regulated via an initiator-like element in their proximal promoters. To determine the elements that contribute to the gastrointestinal expression of UGT1A8 and -1A10, we conducted deletion analysis of the UGT1A8, -1A9, and -1A10 promoters in the colon-derived cell line Caco2. DNA elements contributing significantly to UGT1A8, -1A9, and -1A10 promoter activity were found to reside primarily within 140 base pairs of the transcription start site. Within this region, putative binding sites for the intestine-specific transcription factor, caudal-related homeodomain protein 2 (Cdx2), and hepatocyte nuclear factor 1 (HNF1) were identified. Using gel shift and functional assays, HNF1alpha was demonstrated to bind to and activate the UGT1A8, -1A9, and -1A10 promoters. In contrast, Cdx2 bound to and activated the UGT1A8 and -1A10 promoters but could not activate the UGT1A9 promoter. A single base pair difference between the UGT1A8 and -1A10 promoters, three base pairs downstream of the consensus Cdx2 site, contributed to the observed difference in Cdx2 binding and Cdx2-mediated promoter activation of these two promoters. In addition, Cdx2 was shown to cooperate with HNF1alpha to synergistically activate the UGT1A8, -1A9, and -1A10 promoters. These studies provide insight into the mechanisms controlling the extrahepatic expression of the UGT1A8, -1A9, and -1A10 genes.