It is worth investigating heterotypic cell-cell interactions by mimicking their in vivo structures and environment. In the present study, physiological cellular response and behavior of hepatocytes and endothelial cells were investigated by controlling their contact periphery in a new co-culture system. Rat primary hepatocytes and bovine endothelial cells were co-cultured on a dually patterned surface. Hepatic physiological functions such as albumin secretion and ammonium metabolism were enhanced by increasing heterotypic cell-cell interactions in a patterned co-culture. Furthermore, enhanced hepatic functions through heterotypic interactions are effective within a limited area apart from endothelial cells as evidenced by immunofluorescence staining of hepatic intracellular albumin, indicating that heterotypic interactions act in a paracrine manner. Thus, heterotypic cell communications that play indispensable roles in increasing hepatic physiological functions should be obtained with an increasing periphery of two-cell domains. These findings are important for the reconstruction of complex tissues such as liver and pancreas. (c) 2006 Elsevier Inc. All rights reserved.