Prolactin participates in the regulation of liver function. However, prolactin receptor (PrlR) expression and its regulation have been described only for hepatocytes. In this study, we investigated the expression and regulation of PrlR isoforms in the other important intrahepatic cellular compartment: the biliary epithelial cells, or cholangiocytes. Our aim was to determine whether prolactin should be considered as a potential regulator of cholangiocyte function under normal and pathological conditions. Cholangiocytes and hepatocytes were differentially isolated from rat liver. PrlR expression was analysed at the mRNA level by isoform-specific semiquantitative PCR, and at the protein level by immunostaining of liver sections. Hormonal regulation of PrlR expression was evaluated by comparing intact rats with gonadectomized, pituitary-grafted or bromocriptine-treated animals. Common bile-duct ligation was used as the experimental model of cholestasis. Our results demonstrate that the expression pattern and regulation of PrlR isoforms is totally different in cholangiocytes compared with hepatocytes: (1) mature rat cholangiocytes express low levels of PrlR, while it is very high in hepatocytes, (2) only the long isoform is detected in cholangiocytes, while the short isoform predominates in hepatocytes and (3) PrlR levels in cholangiocytes are induced by obstructive cholestasis, but not by sex hormones or prolactin, while it is the opposite in hepatocytes. From these data, the actions of prolactin on liver are anticipated to exhibit strong cell-type specificity in both normal and pathological conditions.