Despite the success of protease inhibitor (PI)-based antiretroviral therapy, hepatotoxicity has been associated with the concomitant use of PIs. Inhibition of transport proteins by combined administration of PIs may enhance hepatocyte accumulation of drugs and/or endogenous bile acids (BAs), increasing the risk of drug-induced liver injury (DILI). This study was designed to examine the effects of two frequently used PIs, lopinavir (LPV) and ritonavir (RTV), alone and in combination, on: (1) hepatic transport of the model BAs taurocholate (TC) and chenodeoxycholate (CDCA) during acute (10 min) exposure, and (2) hepatobiliary disposition of endogenous BAs after 24-hr exposure. Studies were conducted in day 4 rat sandwich-cultured hepatocytes (SCH) seeded on 24- or 6-well plates. For transport studies, [3H]-TC and [14C]-CDCA (1 μM, 10 min, n=3) accumulation in cells+bile and cells was determined during co-incubation with LPV (5-50 μM), with and without RTV (5 μM), using B-CLEAR® technology. The TC biliary excretion index (BEI), which represents the fraction of accumulated substrate in the bile compartment, was determined. Treatment with LPV or RTV (5 μM) decreased the TC BEI by 29% and 70%, respectively, and the CDCA BEI by 86% and 88%, respectively. LPV concentrations exceeding 10 μM ablated TC biliary excretion, irrespective of RTV co-incubation. Cellular TC accumulation was increased by 10 and 25 μM LPV, and further increased when combined with RTV. Cellular CDCA accumulation was increased by LPV (5-50 μM), and further increased in the presence of RTV. In separate studies, SCH were incubated for 24 hr with LPV (5 or 50 µM) or RTV (5 μM), alone and in combination, and cellular concentrations of endogenous BAs (TC, taurochenodeoxycholate, glycocholate and glycochenodeoxycholate) were quantified by LC/MS/MS. Cellular TC concentrations were decreased from control values of 4.79 to 1.65 and 0.86 pmol/mg protein in the presence of RTV and RTV+LPV (5 µM), respectively. Cellular taurochenodeoxycholate concentrations were decreased from control values of 1.65 to 0.72 and 0.31 pmol/mg protein in the presence of RTV and RTV+LPV (5 µM), respectively. Co-incubation of RTV with 5 or 50 µM LPV decreased total BA cellular concentrations from 6.98 to 1.17 and 0.95 pmol/mg protein, respectively; cellular glycocholate and glycochenodeoxycholate concentrations were negligible. In conclusion, acute (10 min) PI exposure increased cellular BA accumulation; conversely, prolonged (24 hr) PI exposure decreased total BA cellular concentrations. The SCH model is a useful tool to evaluate the effects of drugs on BA disposition. This system enables direct quantification of cellular concentrations of endogenous BAs, and may aid in predicting risk of DILI. Supported by NIH GM41935.