The bifunctional hepatic protein, microsomal epoxide hydrolase (mEH), plays a central role in the metabolism of many xeno-biotics as well as mediating the Na+-dependent uptake of bile acids in parallel with the Na+-taurocholate co-transporting protein (ntcp). Previous studies have established that mEH is expressed in the endoplasmic reticulum with two topological orientations, where the type II form is targeted to the plasma membrane. In this report the topology and transport properties of mEH as a function of plasma membrane expression in cultured hepatocytes, transfected Madin-Darby canine kidney cells expressing mEH (MDCK[mEH]), and the human hepatoma cell line, HepG2, were studied using confocal fluorescence microscopy and substrate uptake measurements. Analysis of mEH localization with an anti-mEH monoclonal antibody demonstrated the expression of one topological for m on the plasma membrane of hepatocytes and MDCK[mEH] cells where both systems exhibited Na+-dependent bile acid uptake. In contrast, Na+-dependent bile acid transport in HepG2 cells and hepatocytes in culture (72h) was substantially reduced as was the expression of ntcp. Although the total mEH level was undiminished, the decrease of bile acid transport was associated with the loss of mEH surface expression possibly resulting from an alteration in mEH endoplasmic reticulum topology and/or the plasma membrane protein targeting system in these de-differentiated cells. (C) 2003 Elsevier Inc. All rights reserved.