In vivo, proteins of the hepatocyte plasma membrane are asymmetrically distributed, making it possible to distinguish a sinusoidal, a lateral and a canalicular domain. The conditions that determine hepatocyte plasma membrane polarity have been investigated in vitro, using three monoclonal antibodies directed against integral membrane proteins, which were characteristic of each domain. The localization of the three antigens was studied by immunolabelling of hepatocytes isolated from adult rat liver, primary monolayer cultures and rat hepatoma cell lines. When hepatocytes were isolated, the three antigens spread over the entire cell surface. The lateral antigen redistributed at lateral sites as soon as cell-cell contacts were established, 4h after the beginning of primary culture. The sinusoidal and canalicular antigens became asymmetrically distributed after 48 h of primary culture, after the formation of bile canaliculus-like structures. In most of the hepatoma lines studied, the three antigens were expressed, except that the canalicular antigen was fully expressed in differentiated clones only. The lateral antigen was always distributed on thecontiguous membranes of clustered hepatoma cells, whereas the sinusoidal and canalicular antigens were localized on the entire plasma membrane. However, in a few cells of some clones in which bile canaliculus-like structures were observed, the canalicular membranes were strongly labelled only with the canalicular antibody. In the absence of bile canalicular formations, in both primary culture and cell lines, the canalicular antigen and, to a lesser extent, the sinusoidal antigen accumulated in the Golgi apparatus, suggesting that their transport to the cell surface was altered in the absence of a bile pole. These results show that in hepatic cells, polarization of the plasma membrane is determined by: (1) the existence of cell-cell contacts, which is correlated with the domain-specific localization of the lateral antigen; and (2) the formation of bile canaliculi, which would trigger the development of an asymmetrical distribution of the sinusoidal and canalicular antigens.