Intrahepatic bile acid accumulation due to inhibition of the bile salt export pump (BSEP) has been proposed as a mechanism for drug-induced cholestasis. Many cholestatic drugs do not initiate hepatotoxicity in rats, although they inhibit rat Bsep and cause elevated serum bile acid concentration. In this study, we examined changes in the taurocholate (TC) transport in response to cholestatic drug treatments in human and rat sandwich-cultured hepatocytes. Our experimental setup allows studying the basolateral and canalicular efflux simultaneously, thus comparing drug-induced changes in the vectorial efflux of TC. We found that TC elimination highly differs in human and rat hepatocytes. In human hepatocytes, an equal fraction of TC(uptake) was eliminated by basolateral (34.8%) and canalicular (34.4%) transporters and remained in the cells (30.5%), while in the case of rats, the basolateral transport was dominant (71.7%) and intracellular TC accumulation was negligible (6.9%). The inhibition of BSEP/Bsep resulted in significantly higher intracellular TC(conc) in humans than in rats. The 15-fold difference in intracellular TC(conc) of control in human versus rat hepatocytes was increased 25-fold by troglitazone treatment. MK571 and indomethacin decreased the basolateral efflux and significantly increased the intracellular TC(conc) in rats. In rat hepatocytes, the highest intracellular TC(conc) was observed with cyclosporine A and glibenclamide, which inhibited TC elimination in both directions. Nevertheless, the basolateral transport remained dominant. We conclude that in rats, the higher rate of basolateral bile salt efflux represents an additional protective mechanism in cholestasis, which contributes to species differences in response to hepatotoxic drugs.