1. The effects of HIV protease inhibitors (PI) on accumulation of the fluorescent bile salt analogue cholyl-glycylamido-fluorescein (CGamF) were determined in OATP1B1- and 1B3-expressing CHO cells. In addition, interaction studies in Caco-2 monolayers, known to only express the OATP2B1 isoform, were conducted using the established OATP substrate estrone-3-sulfate (E3S), since no CGamF accumulation was observed in Caco-2 monolayers. 2. CGamF appeared an excellent substrate for the OATP1B subfamily, with net accumulation clearance values of 7.8 and 142 !l/min/mg protein in 1B1 and 1B3-transfected cells, respectively. Ki values reflecting inhibition of CGamF accumulation by HIV PI correlated well between OATP1B1 and 1B3-expressing cells. Lopinavir was the most potent inhibitor (Ki 0.5-1.4 !M) of OATP1B-mediated CGamF accumulation, compared to atazanavir, darunavir, ritonavir and saquinavir (Ki between 1.4 and 3.3 !M). 3. Inhibitory profiles towards OATP2B1-mediated E3S accumulation were different with only indinavir, saquinavir and ritonavir showing substantial effects. 4. In conclusion, OATP1B3 appears to be a major transport mechanism mediating sodium-independent CGamF accumulation in human liver and CGamF could be used as a probe substrate for in vitro drug interaction studies. The remarkably potent inhibition of OATP1B1 by lopinavir may explain some clinically relevant drug interactions between lopinavir and OATP1B substrates such as fexofenadine.