This study was undertaken to examine the influence of seeding density, extracellular matrix and days in culture on bile acid transport proteins and hepatobiliary disposition of the model bile acid taurocholate. Mouse hepatocytes were cultured in a sandwich configuration on six-well Biocoat plates with an overlay of Matrigel (BC/MG) or gelled-collagen (BC/GC) for 3 or 4 days at seeding densities of 1.0, 1.25, or 1.5 x 10(6) cells/well. The lower seeding densities of 1.0 and 1.25 x 10(6) cells/well resulted in good hepatocyte morphology and bile canalicular network formation, as visualized by 5-(and 6)-carboxy-2',7'-dichlorofluorescein accumulation. In general, taurocholate cellular accumulation tended to increase as a function of seeding density in BC/GC; cellular accumulation was significantly increased in hepatocytes cultured in BC/MG compared to BC/GC at the same seeding density on both days 3 and 4 of culture. In general, in vitro intrinsic biliary clearance of taurocholate was increased at higher seeding densities. Levels of bile acid transport proteins on days 3 and 4 were not markedly influenced by seeding density or extracellular matrix except for multidrug resistance protein 4 (Mrp4), which was inversely related to seeding density. Mrp4 levels decreased approximately 2- to 3-fold between seeding densities of 1.0 x 10(6) and 1.25 x 10(6) cells/well regardless of extracellular matrix; an additional approximately 3- to 5-fold decrease in Mrp4 protein was noted in BC/GC between seeding densities of 1.25 x 10(6) and 1.5 x 10(6) cells/well. Results suggest that seeding density, extracellular matrix and days in culture profoundly influence Mrp4 expression in sandwich-cultured mouse hepatocytes. Primary mouse hepatocytes seeded in a BC/MG configuration at densities of 1.25 x 10(6) cells/well and 1.0 x 10(6), and cultured for 3 days, yielded optimal transport based on the probes studied. This work demonstrates the applicability of the sandwich-cultured model to mouse hepatocytes.