Transporters for organic anions play an important role in the excretion of a variety compounds by the kidney. Many antivirals used for treatment of HIV infection or chronic hepatitis B may be excreted via the kidney by human organic anion transporter 1 (hOAT1) located characteristically in the proximal renal tubules. Concomitant administration of antivirals with drugs from other therapeutic groups (e. g. antifungals, antibiotics) may lead to potential interactions with this transport system. Such interactions in the kidney may result in significant changes in the renal excretion rate of the interacting compounds. The aim of this study was to compare the affinity of three antiviral drugs (tenofovir, elvitegravir, entecavir) and several potentially concomitantly used drugs (amphotericin B, gentamicin) to hOAT1 using a human cellular in vitro model and evaluate possible transport interactions of the studied drugs at this transport system. Human cervical cancer cell line (HeLa) transiently transfected with hOAT1 was used for the experiments. A possible positive or negative impact of the interactions of the tested drugs at hOAT1 on renal cytotoxicity was tested in human renal cell line HK-2. The used HeLahOAT1 cell model showed probenecid-sensitive uptake of [3H]para-aminohippuric acid, a prototypical hOAT1 substrate. In addition, temperature-dependent accumulation of [3H]tenofovir in HeLahOAT1 cells was demonstrated. These findings confirmed proper functioning of hOAT1 in HeLahOAT1 cells. In the affinity transport studies, the rate of inhibition of intracellular accumulation of [3H]para-aminohippuric acid was induced by gradually increasing concentration of the tested compound. The found values for affinity to hOAT1 can be ranged from the highest to the lowest affinity: amphotericin B > entecavir > tenofovir >> elvitegravir. An interaction of gentamicin at hOAT1 was not proved using available incubation concentrations. Due to the very high affinity to the hOAT1, amphotericin B was selected for interaction experiment with [3H]tenofovir. A significant interaction at hOAT1 between these two compounds was demonstrated. Inhibition of [3H]tenofovir transport by amphotericin B was stronger than that of [3H]para-aminohippuric acid. Although efficiently transported by hOAT1, tenofovir showed lower cytotoxic effect than amphotericin B in HK-2 cells. In conclusion, the study confirms that hOAT1 may contribute to the influx of all three tested antiviral drugs into cells. However, the rate of transport by hOAT1 will be various due to the significant differences in the affinity to the transporter. The found competition of amphotericin B and tenofovir at hOAT1 may potentially change renal excretion parameters of the competitors during concomitant administration. The study was supported by Charles University in Prague (Project SVV 263003), grant GAUK No. 360811/FaF/C-LEK and grant IGA MZ No. NT12398-4/2011.