The rat UDP glucuronosyltransferase UGT2B1 is expressed mainly in the liver where it glucuronidates steroids and environmental toxins and carcinogens. A region between -42 and -55 bp upstream from the UGT2B1 gene transcription start site was previously identified as sharing sequence similarity with the hepatocyte nuclear factor 1 (HNF1) consensus binding site. In this study, the importance of this region in the regulation of the UGT2B1 gene was confirmed by functional and DNA binding assays. A minimal UGT2B1 gene promoter containing the putative HNF1 binding site was fused to the CAT reporter gene and transfected into HepG2 cells. Only low levels of CAT activity were detected. This activity was increased 50-fold when an HNF1α expression vector was co-transfected with the UGT2B1 promoter CAT construct but was not altered when a HNF1β expression vector was used. A UGT2B1 promoter construct with the HNF1-like region deleted was not activated by either co-transfected HNF1 expression vector. DNase 1 footprinting and gel-shift analysis demonstrated that nuclear proteins present in both HepG2 cells and rat liver bind to the HNF1-like element. The presence of HNF1α in these nuclear proteins that bind to the HNF1-like element was confirmed by supershift analysis with antisera to HNF1α. Specific binding of nuclear proteins to the HNF1-like element was not seen in extracts from three cell lines derived from nonhepatic tissues. These data strongly suggest that the liver-enriched factor HNF1α binds to, and activates, the UGT2B1 gene promoter.