Depending on their position within the liver lobule, hepatocytes fulfill different metabolic functions. Cytochrome P450 (CYP) 2E1 is a drug-metabolizing enzyme which is exclusively expressed in hepatocytes surrounding branches of the hepatic central vein. Previous publications have shown that signaling through the Wnt/beta-catenin pathway, a major determinant of liver zonation, and the hepatocyte-enriched transcription factor HNF (hepatocyte nuclear factor) 1alpha participate in the regulation of the gene. This study was aimed to decipher the molecular mechanisms by which the two transcription factors, beta-catenin and HNF1alpha, jointly regulate CYP2E1 at the gene promoter level. Chromatin immunoprecipitation identified a conserved Wnt/beta-catenin-responsive site (WRE) in the murine Cyp2e1 promoter adjacent to a known HNF1alpha response element (HNF1-RE). In vitro analyses demonstrated that both, activated beta-catenin and HNF1alpha, are needed for the full response of the promoter. The WRE was dispensable for beta-catenin-mediated effects on the Cyp2e1 promoter, while activity of beta-catenin was integrated into the promoter response via the HNF1-RE. Physical interaction of beta-catenin and HNF1alpha was demonstrated by co-immunoprecipitation. In conclusion, present data the first time identify and characterize the interplay of HNF1alpha and beta-catenin and elucidate molecular determinants of CYP2E1 expression in the liver.