Bile salts are predominantly taken up by hepatocytes via the basolateral Na⁺ taurocholate co-transporting polypeptide (NTCP/SLC10A1) and secreted into the bile by the bile salt export pump (BSEP/ABCB11). In the present study, we transfected rat Ntcp and rat Bsep into polarized Madin-Darby canine kidney cells and characterized the transport properties of these cells for eight bile salts. Immunohistochemical staining demonstrated that Ntcp was expressed at the basolateral domains whereas Bsep was expressed at the apical domains. Basal-to-apical transport of taurocholate across the monolayer expressing only Ntcp and that co-expressing Ntcp/Bsep was observed, whereas the flux across the monolayer of control and Bsep expressing cells was symmetrical. Basal-to-apical transport of taurocholate across Ntcp/Bsep co-expressing monolayers was significantly higher than that across monolayers expressing only Ntcp. Kinetic analysis of this vectorial transport of taurocholate gave an apparent K_m value of 13.9 ± 4.7 µM for cells expressing Ntcp alone which is comparable to 22.2 ± 4.5 µM for cells expressing both Ntcp and Bsep and V_max values of 15.8 ± 4.2 and 60.8 ± 9.0 pmol/min/mg protein for Ntcp alone and Ntcp and Bsep co-expressing cells, respectively. Transcellular transport of cholate, glycocholate, taurochenodeoxycholate, chenodeoxycholate, glycochenodeoxycholate, tauroursodeoxycholate, ursodeoxycholate, and glycoursodeoxycholate, but not that of lithocholate, was also observed across the double transfectant. This double expressing system can be used as a model to clarify vectorial transport of bile salts across hepatocytes under physiological conditions.