Rat hepatocytes cultured in a sandwich configuration form functional canalicular networks. The influence of extracellular matrix configuration, medium composition, and confluency on the expression and function of Bsep, Mrp2, and Mdr1a/b in sandwich-cultured (SC) rat hepatocytes was examined. Primary rat hepatocytes were: (1) maintained in various extracellular matrix sandwich configurations, (2) cultured in Dulbecco's modified Eagle's medium (DMEM), Modified Chee's medium (MCM) or Williams' E medium (WME), and/or (3) plated at decreasing cell density. Bsep, Mrp2, and Mrdr1a/b expression in day 4 SC rat hepatocytes was assessed by Western blot; function was measured by accumulation of taurocholate, 5(and 6)-carboxy-2′,7′-dichlorofluorescein, and rhodamine 123, respectively, in canalicular networks. In general, the extracellular matrix conditions examined resulted in similar protein expression and function. Function of Bsep, Mrp2, and Mdr1a/b was higher in SC rat hepatocytes maintained in DMEM or WME. Mrp2 and Mdr1a/b expression, representative of total cellular content, did not always correlate directly with function, which should be reflective of canalicular membrane expression. Mrp2 expression decreased significantly as cell density decreased in SC hepatocytes. Low plating density in Biocoat™ plates resulted in poor canalicular network formation and reduced function of Mrp2 and Mdr1a/b. Expression and/or function of Mrp2 and Mdr1a/b in rat hepatocytes cultured in a sandwich configuration may be influenced by plating density and media type.