Sodium taurocholate co-transporting polypeptide (Ntcp) is the major uptake system for conjugated bile acids. Deletions of HNF1 and RXR:RAR binding sites in the mouse 5'-flanking region corresponding to putatively central regulatory elements of rat Ntcp do not significantly reduce promoter activity. We hypothesized that HNF4, which is increasingly recognized as a central regulator of hepatocyte function, may directly transactivate mouse Ntcp. A 1.1 kb 5'-upstream region including the mouse Ntcp promoter was cloned and compared to the rat promoter. In contrast to a moderate 3.5-fold activation of mNtcp by HNF1, HNF4 cotransfection led to a robust 20-fold activation. Deletion analysis of mouse and rat Ntcp promoters mapped a conserved HNF4 consensus site at -345/-326 and -335/-316 bps, respectively. p-475bpmNtcpLUC is not transactivated by HNF1 but shows a 50-fold enhanced activity upon cotransfection with HNF4. Gel mobility shift assays demonstrated a complex of the HNF4-element formed with liver nuclear extracts which was blocked by a HNF4 specific antibody. HNF4 binding was confirmed by chromatin immunoprecipitation. Using Hepa 1-6 cells HNF4-knockdown resulted in a significant 95% reduction in NTCP mRNA. In conclusion, mouse Ntcp is regulated by HNF4 via a conserved distal cis-element independently of HNF1.