The cell-basement membrane interaction is an important determinant of epithelial cell polarity. Although hepatocytes in situ are polarized, no morphologically identifiable basement membrane is found at their basal surface. However, several studies have demonstrated immunoreactivity to basement membrane proteins in the space of Disse, indicating the existence of an extracellular matrix, albeit of low density. Therefore we hypothesized that the interaction of hepatocytes with this matrix may determine their polarity and asked whether a basement membrane-like substrate could reestablish hepatocyte polarity in vitro. For this purpose, established monolayers of primary rat hepatocytes were cultured overlaid with a basement membrane-like matrix extracted from the Engelbreth-Holm-Swarm mouse tumor, mimicking thus the in situ tissue architecture. The hepatocytes in this culture configuration, unlike hepatocytes in classic cultures, developed distinct membrane domains, as demonstrated by the reformation of gamma-glutamyltranspeptidase, Mg(2+)-ATPase-positive bile canalicular networks and intercellular gap junctions immunolocalized to the lateral membrane with antibodies to connexin 32. The actin cytoskeleton of these cells reorganized into pericanalicular webs, and no accumulation of "stress" filaments was found beneath the membrane facing the medium. Golgi complexes appeared to be preferentially located in mitochondria-poor pericanalicular cytoplasm, indicating the polarized distribution of these organelles. Together, these data indicate that a basement membrane-like substrate present between hepatocytes and nutrient medium restores the polarity of these cells in culture. Extrapolation of these findings to the intact liver suggests that the matrix in Disse's space governs the development of hepatocyte polarity.