Organic anion transporting polypeptide (oatp1) has been cloned from rat liver as one of the transporters responsible for the hepatic uptake of ligands, and its substrate specificity has been determined. However, the contribution of oatp1 to the Na+-independent uptake of ligands into rat hepatocytes remains to be investigated. In the present study, we determined the contribution of oatp1 and examined the uptake of ligands into primary cultured hepatocytes (cultured for 4 h) and into COS-7 cells transiently expressing oatp1 and normalized using estradiol-17beta-D-glucuronide as a reference compound. Western blot analysis indicated that oatp1 was less extensively glycosylated in transfected COS-7 cells, and the expression level in transfectant was one-seventh that in rat liver. The Km values for the uptake of estradiol-17beta-D-glucuronide were similar for cultured hepatocytes and oatp1-transfected COS-7 cells (Km = 12.3 versus 20.4 microM), although the Vmax value for oatp1-transfected COS-7 cells was one-seventh that for cultured hepatocytes (Vmax = 1.30 versus 0.175 nmol/min/mg protein). The contribution of oatp1 to the Na+-independent uptake of taurocholic acid and cholic acid into rat hepatocytes was more than 50 to 60%, whereas the corresponding values for the sulfate-conjugates of estrone and 6-hydroxy-5, 7-dimethyl-2-methylamino-4-(3-pyridylmethyl)benzothiazole were 20 to 30%. In addition, the analysis indicated that the contribution of oatp1 to the Na+-independent uptake of several ligands [glucuronide-conjugate of 6-hydroxy-5, 7-dimethyl2-methylamino-4-(3-pyridylmethyl)benzothiazole, ibuprofen, pravastatin, ouabain, and 2,4-dinitrophenyl-S-glutathione] was minimal. Collectively, the transfected COS-7 cells may be used to quantitatively predict oatp1 activity in hepatocytes after correction of its expressed amount. It is also suggested that multiple transport mechanisms are responsible for the Na+-independent uptake of organic anions into hepatocytes.