UDP-glucuronosyltransferase 1A6 (UGT1A6) is a major UGT contributing to the glucuronidation of small phenolic compounds. The gene for rat 1A6 is expressed using two promoters, a distal promoter P1 and a proximal promoter P2. Transcripts from P2 are high in liver, gastrointestinal tract, and kidney, whereas P1 transcripts predominate in other tissues. Here we report evidence for primary control of the P2 promoter by hepatic nuclear factor 1 (HNF1). Transient transfection of a P2 reporter plasmid, p(-1354/+65) 1A6P2-luc, resulted in enhanced luciferase activity in HepG2 but not Hepa1 cells compared to cells transfected with pGL3-Basic control vector. A truncated reporter under the control of -224 to +65 exhibited comparable activity. Footprint analysis of the -224/+65 fragment revealed specific binding by rat liver nuclear protein to a region between bases -60 and -37. The binding activity was also observed with HepG2 cell but not Hepa1 cell extract. Electrophoretic mobility shift assays were consistent with the presence of HNF1 in the binding complexes. The functionality of an HNF1-binding site at -51/-37 is also supported by (1) marked decreases in the activity of P2 reporter plasmids containing a three-base substitution in the proposed HNF1 binding site and (2) the enhancement of P2 reporter activity following cotransfection of an HNF1alpha expression plasmid. The UGT1A6 P1 promoter lacks an HNF1 binding site in the analogous position and showed little response to HNF1 overexpression. Although these data do not strictly rule out an interaction between the P1 promoter and HNF1 bound to -51/-37 of P2, the results suggest a mechanism for the more abundant expression of P2-derived UGT1A6 transcripts in liver and other HNF1-enriched tissues.