The hepatic uptake of bile acids from the portal circulation across the sinusoidal plasma membrane is primarily mediated by the sodium-dependent bile acid cotransporter, the Ntcp protein. Hepatic sodium-dependent bile acid uptake is developmentally-regulated, and Ntcp mRNA and protein levels in the developing rat are principally driven by mRNA transcription rates [J. Biol. Chem. 270:20841-6, 1995]. Significant Ntcp mRNA levels are demonstrable by fetal day 21, with adult levels apparent by 1 week of life. The factors determining the developmental expression of Ntcp mRNA transcription are unknown. The AIM of our study was to determine the developmental expression of two rat nuclear factors recently shown to regulate the basal expression of the rat Ntcp promoter [J. Biol. Chem. 271:15211 - 15221, 1996]. Methods: Crude rat liver nuclear extracts were prepared from fetal days 18, 20, 22 and postnatal days 1, 3, 7 as well as from adult rats. DNase I footprinting and gel mobility shift assays were performed using standard techniques. Results: Footprint analysis of the rat Ntcp promoter revealed two protein-bound regions, one determined to be a hepatocyte nuclear factor 1 site (HNF1; nt -7 to +4) and the other an unknown protein bound to the footprint B site [nt -50 to -37]. Gel mobility shift assays provided evidence of HNF1 binding from fetal day 22 onward, with a gradual postnatal increase in intensity to adult levels by 1 week of life. Interestingly, the HNF1 complex migrated at a distinctly slower mobility using adult liver nuclear extracts than from any other age group, suggesting the formation of a macromolecular complex in adult rat liver nuclei. Gel mobility shift assays of the footprint B site revealed the appearance of a specifically-bound doublet from nuclei of each age group after fetal day 22, with an abrupt increase in intensity after 1 week of life. Summary and Conclusions: Both HNF1 and an unknown Ntcp footprint B binding protein bind to their respective sites in the rat Ntcp promoter prior to birth, suggesting that they play an important role in the developmental expression of the rat Ntcp gene. The expression of these transcriptional regulators coincides with the timing of the initiation of Ntcp mRNA transcription.