Transport systems involved in uptake and biliary secretion of bile salts have been extensively studied in rat liver; however, little is known about these systems in the human liver. In this study, we investigated taurocholate (TC) transport in canalicular and basolateral plasma membrane vesicles isolated from 15 human livers (donor age 6–64 yr). ATP stimulated the uptake of TC into both canalicular and basolateral human liver plasma membrane vesicles (cLPM and bILPM, respectively). Considerable interindividual variations in the transport velocity were observed in the different membrane preparations used: 9.0 ± 1.3 (mean ± SEM, n = 17; range 1.6–18.0) and 9.3 ± 2.0 (range 1.1–29.8) pmol TC · protein−1 · min−1 at 1.0 μM TC for cLPM and bILPM, respectively. TC transport was temperature sensitive and showed saturation kinetics with a high affinity for TC (Km 4.2 ± 0.7 μM and 3.7 ± 0.5 μM for cLPM and bILPM, respectively). Transport was dependent on the ATP concentration and saturable (Km 0.25 ± 0.03 mM, n = 3). Neither nitrate, which reduces membrane potential, nor the protonophore FCCP strongly inhibited ATP-dependent TC transport, indicating that membrane potential and proton gradient are not involved in this process. TC transport was significantly inhibited by the classical anion transport inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonate (250 μM) and the glutathione conjugate S-(2,4-dinitrophenyl)glutathione (100 μM). In conclusion, high affinity ATP-dependent TC transport is present in human liver at both the canalicular and the basolateral sides of the hepatocyte.