The human UDP glucuronosyltransferase UGT2B17, glucuronidates androgens and is expressed in the liver and the prostate. Although evidence suggests that variations in UGT2B17 expression between tissues may be a critical determinant of androgen response, the factors that regulate UGT2B17 expression in the liver and prostate are unknown. In this study, we have isolated a 596 bp promoter of the UGT2B17 gene and studied its regulation in the liver cell line, HepG2 and the prostate cell line, LNCaP. The transcription start site of UGT2B17 was mapped and proteins that bound to the proximal promoter were detected by DNase1 footprint analysis. A region (-40 to -52 bp) which resembled a hepatocyte nuclear factor 1 (HNF1) binding site bound proteins in nuclear extracts from HepG2 cells, but did not bind proteins from LNCaP nuclear extracts. In HepG2 cells, HNF1alpha bound to this region and activated the UGT2B17 promoter, as assessed by functional and gel shift assays. HNF1alpha activation of the promoter was prevented by mutation or deletion of the putative HNF1 site. The related transcription factor HNF1beta, which is present in HepG2 cells, did not activate the promoter. The UGT2B17 promoter could also be activated by exogenous HNF1alpha in LNCaP cells. However, because these cells do not contain HNF1alpha, other transcription factors must regulate the UGT2B17 promoter. Cotransfection experiments showed that HNF1beta, elevates promoter activity in LNCaP cells. This activation did not involve the putative HNF1 region (-40 to -52 bp) since mutation of this region did not affect promoter activation by HNF1beta. These results suggest that the UGT2B17 promoter is regulated by different factors in liver-derived HepG2 and prostate-derived LNCaP cells.