Na(+)/taurocholate cotransporting polypeptide (ntcp) mediates the uptake of bile salts from plasma across the basolateral domain of the hepatocyte. We have demonstrated that ntcp expression can be induced by prolactin (PRL) and placental lactogen via the PRL receptor and signal transducers and activators of transcription (Stat)5a pathway. However, elevated levels of placental lactogen do not increase the expression of ntcp in pregnant rats. Because plasma estradiol (E(2)) levels are also elevated in pregnancy, we investigated the inhibitory effects of E(2) on PRL-induced ntcp activation. E(2) treatment inhibited the PRL-induced increase in liver ntcp mRNA to the same levels as in rats treated with E(2) alone. Estrogen receptor-alpha (ERalpha) mRNA and protein expression in liver were increased 2.6-fold and 2.2-fold, respectively, in pregnancy relative to controls. In HepG2 cells, E(2) repressed PRL-induced ntcp reporter gene expression in a dose-dependent manner in the presence of cotransfected ERalpha. The ERalpha antagonist ICI 182,780 reversed E(2)-induced repression, indicating specificity of inhibition by E(2). Overexpression of coactivator p300 did not reverse the inhibitory effects of E(2) and ERalpha. Western and gel shift analysis revealed that E(2)-bound ERalpha decreased the tyrosine phosphorylation and DNA-binding activity of Stat5a, indicating that the inhibitory effect of E(2) was mediated, at least in part, by interfering with PRL-mediated signal transduction. The present studies demonstrate the physiological significance of cross-talk between ERalpha and Stat5a in liver, in which both proteins are expressed. These data also establish a novel mechanism by which expression of ntcp, an important hepatic bile acid transporter, can be regulated by multiple hormones.