Сасо-2 cell line is widely used in in vitro studies of the intestinal wall permeability for drugs, but transport activity of these cells has not been thoroughly studied. We analyzed localization and expression of nucleoside transporters of the ENT family transferring a number of anticancer and antiviral drugs. Immunocytochemical staining revealed apical localization of ENT1 and integral expression of ENT2 in Caco-2 cells. Based on these data and on the value of hypoxanthine uptake by these cells, we created a mathematical model allowing quantification of transporter expression on the apical and basolateral membranes of Caco-2 cells. The discrepancy between gene and protein expression of the transporter complicating prediction of patient’s sensitivity to the drug upon individual therapy selection is discussed.