The hepatobiliary disposition of xenobiotics may involve passive and/or active uptake, metabolism by cytochromes P450, and excretion of the parent compound and/or metabolite(s) into bile. Although in vitro systems have been used to evaluate these individual processes discretely, mechanistic in vitro studies of the sequential processes of uptake, metabolism, and biliary or basolateral excretion are limited. The current studies used sandwichcultured (SC) rat hepatocytes combined with a comprehensive pharmacokinetic modeling approach to investigate the hepatobiliary disposition of terfenadine and fexofenadine, a model drug/ metabolite pair. The metabolism of terfenadine and the biliary excretion of terfenadine and fexofenadine were determined in control and dexamethasone-treated SC rat hepatocytes. Dexamethasone (DEX) treatment increased the formation rates of the terfenadine metabolites azacyclonol and fexofenadine ϳ20-and 2-fold, respectively. The biliary excretion index (BEI) of fexofenadine, when generated by terfenadine metabolism, was not significantly different from the BEI of preformed fexofenadine (15 Ϯ 2% versus 19 Ϯ 2%, respectively). Pharmacokinetic modeling revealed that the rate constant for hepatocyte uptake was faster for terfenadine compared with preformed fexofenadine (2.5 versus 0.08 h Ϫ1 , respectively), whereas the biliary excretion rate constant for preformed fexofenadine exceeded that of terfenadine (0.44 versus 0.039 h Ϫ1 , respectively). Interestingly, the rate constants for basolateral excretion of terfenadine and fexofenadine were comparable (3.2 versus 1.9 h Ϫ1 , respectively) and increased only slightly with DEX treatment. These studies demonstrate the utility of the SC hepatocyte model, coupled with pharmacokinetic modeling, to evaluate the hepatobiliary disposition of generated metabolites.Biliary clearance is a predominant route of elimination for many compounds, including bile salts, organic anions, and organic cations. In recent years, the mechanisms involved in the hepatic uptake, as well as the sinusoidal and canalicular excretion, of many substances have been elucidated. After transport into the hepatocyte, many compounds undergo phase I and/or phase II metabolism before excretion into bile (by canalicular transport proteins) or sinusoidal blood (by basolateral transport proteins). Pang and coworkers have clearly established that, in some cases, the hepatic disposition of an administered preformed metabolite may be very different from a metabolite generated in the intact organ (Pang et al., 1984;Tirona and Pang, 1996). However, differences in disposition of a generated versus preformed metabolite are by no means assured and must be demonstrated on a case-by-case basis.Recent advances in molecular biology have allowed the mechanistic investigation of discrete transport or metabolic processes in isolation through administration of parent drug or metabolite to a variety of transfected, cell-based expression systems (Wrighton et al., 1995;Mizuno et al., 2003). Cummins et al....