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 theUGT2B17gene and studied its regulation in the liver cell line, HepG2 and the prostate cell line, LNCaP. The transcription start site ofUGT2B17was 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, HNF1α bound to this region and activated theUGT2B17promoter, as assessed by functional and gel shift assays. HNF1α activation of the promoter was prevented by mutation or deletion of the putative HNF1 site. The related transcription factor HNF1β, which is present in HepG2 cells, did not activate the promoter. TheUGT2B17promoter could also be activated by exogenous HNF1α in LNCaP cells. However, because these cells do not contain HNF1α, other transcription factors must regulate theUGT2B17promoter. Cotransfection experiments showed that HNF1β 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 HNF1β. These results suggest that theUGT2B17promoter is regulated by different factors in liver-derived HepG2 and prostate-derived LNCaP cells.