The intracellular mechanism(s) underlying the upregulation of the hepatic Na+/taurocholate cotransporting polypeptide (ntcp) by prolactin (PRL) are unknown. In this report, we demonstrate a time-dependent increase in nuclear translocation of phosphorylated liver Stat5 (a member of the ignal ransducers and ctivators of ranscription family) that correlated with suckling-induced increases in serum PRL levels. In electrophoretic mobility gel shift assays, nuclear Stat5 exhibited specific DNA-binding ability towards IFN-gamma-activated sequence (GAS)-like elements (GLEs; 5'TTC/A-PyNPu-G/TAA-3') located in the -937 to -904 bp region of the ntcp promoter. Transient cotransfections in HepG2 cells revealed that PRL inducibility (2.5-3-fold) required coexpression of the long form of the PRL receptor (PRLRL) and Stat5. Deletion analysis mapped the PRLinducible region to -1237 to -758 bp of the ntcp promoter. Linking this 0.5-kb region to a heterologous thymidine kinase (tk) promoter, or linking multimerized ntcp GLEs either upstream of the ntcp minimal promoter (-158 to +47 bp) or the heterologous promoter conferred dose-dependent PRL responsiveness. The short form of the PRL receptor failed to transactivate ntcp GLEs. These results indicate that PRL acts via the PRLRL to facilitate Stat5 binding to ntcp-GLEs and to transcriptionally regulate ntcp.