To maintain ongoing vectorial bile secretion, hepatocytes localize distinct transport systems at their basolateral and canalicular membrane domains. Here we compare the expression of the basolateral Na+-taurocholate cotransporter (Ntcp) and organic anion transporting polypetides 1 and 2 (Oatp1, Oatp2) and the canalicular bile salt export pump (Bsep) and multidrug resistance–associated protein 2 (Mrp2) in primary cultured rat hepatocytes. During 72 hours of culturing time the messenger RNA (mRNA) and protein levels of Ntcp and Oatp1 decreased in parallel to 2% to 7% of initial values at 3 hours. Although Oatp2 mRNA exhibited a similar down-regulation to 4%, Oatp2 protein and function were maintained at 25% to 47% of initial values. Furthermore, Bsep and Mrp2 protein levels were maintained at about 50%, while the Mrp2 mRNA showed a transient up-regulation to 154% at 24 and 48 hours. Also, induction of Mrp1 mRNA and protein was observed starting after 24 hours. These results indicate transcriptional down-regulation or decreased mRNA stability of Ntcp and Oatp1, transcriptional and posttranslational regulation of Oatp2, Bsep, and Mrp2 and transcriptional up-regulation of Mrp1 in primary cultured hepatocytes. Furthermore, and most importantly, the observed changes in transporter expression closely resemble the altered transporter phenotypes of cholestatic and proliferating hepatocytes in vivo, thus indicating that primary cultured hepatocytes acquire a cholestatic phenotype, and that the transporter expression might be a suitable differentiation marker for maintenance of hepatocytes in vitro.