The uptake of ursodeoxycholic acid (UDCA) was studied in isolated hamster hepatocytes. The uptake was rapid and linear up to 60 seconds for each concentration studied. When the uptake rate was plotted against UDCA concentration, the curve was nonlinear, indicating both saturable and nonsaturable uptake mechanisms. The nonsaturable process had a diffusion constant of 0.01 nmol·s–1·g of cell·μmol/L–1. The saturable component was characterized by a maximum rate of uptake (Vmax) of 5.68 nmol·s–1·g of cell–1 and a Michaelis constant (Km) of 224 μmol/L. In the presence of monensin, ouabain, and amiloride, the uptake of UDCA was significantly decreased by 35% to 55%, whereas the sodium-independent uptake of UDCA was not affected by either monensin or amiloride, thereby confirming sodium dependence of UDCA uptake. The sodium-dependent of UDCA was characterized by a Vmax and a Km of 1.57 nmol·s–1·g of cell–1 and 46 μmol/L, respectively. The rate of uptake of UDCA was maximal at extracellular sodium concentration ≥20 mmol/L. Furthermore, the uptake of UDCA was competitively inhibited by both taurocholic acid and cholic acid with an inhibitory constant (Ki) of 60 μmol/L and 48 μmol/L, respectively. Finally, 1 mmol/L of 4,4′-diisothiocyano-2,2′-disulfonic stilbene (DIDS) inhibited solely the sodium-dependent uptake of cholic acid and UDCA. These findings confirm that the hepatocellular uptake of UDCA involves, at least in part, a sodiumdependent, ouabain, amiloride, and DIDS-sensitive transporter. (Hepatology 1995;21:145–154).