The morphological and cytoskeletal reorganization of collagen-sandwiched rat hepatocytes during the de novo formation of complete canalicular networks was examined by phase, fluorescence and electron microscopy. During the initial stages of membrane repolarization, there was a marked accumulation of both microfilaments and microtubules at the sites of canalicular generation. Microtubule-disrupting agents (colchicine, nocodazole) inhibited the localization of actin filaments at cell margins and the initiation and branching of canalicular networks. After removal of microtubule-disrupting agents, microfilaments relocalized to the canalicular borders and microtubules nucleated along the margins of the bile canaliculi at sites distinct from the peri-canalicular actin networks. Microfilament-perturbing agents (cytochalasin D, phalloidin) did not affect the de novo initiation of bile canaliculi and only slightly impaired the development of canalicular lumina into networks. In established cultures with complete canalicular networks, subsequent treatment with microtubule-disrupting agents did not acutely affect the integrity of preformed canalicular networks. In contrast, treatment with microfilament-perturbing agents caused a marked dilation of most canaliculi. These results illustrate the differential role of the cytoskeleton in the regeneration and maintenance of bile canalicular networks by collagen-sandwiched hepatocytes. Moreover, this study shows the utility of this system as an in vitro model for examining the regulation of cell and membrane polarity.