The hepatocyte plasma membrane presents a morphological and functional regionalization into three domains: the sinusoidal; the lateral, and the canalicular. The mechanisms responsible for the biogenesis and maintenance of this regionalization are poorly understood. In this work, we have used colchicine and phalloidin, two drugs known to interfere with the secretory processes in hepatocytes, to study whether they also affect the transport of membrane proteins. The localization of three plasma membrane antigens was studied by light and electron microscopy using monoclonal antibodies identifying either the sinusoidal (A39) or the lateral (B1) or the canalicular (B10) domains in normal hepatocytes. In rats injected with colchicine (0.25 mg per 100 gm), A39 moved from the sinusoidal membrane to the lateral and canalicular ones, whereas B10 was displaced from the canalicular to the sinusoidal and lateral membranes, resulting after 8 hr in an almost equal labeling of the three domains with both antibodies. In rats injected daily for 7 days with phalloidin (50 micrograms per 100 gm), A 39 became mainly localized on the bile canalicular membrane instead of the sinusoidal one; B10 predominated on the canalicular membrane as in controls but in places it labeled the sinusoidal and lateral domains as well. In bile duct-ligated rats studied for comparison for 4, 10 or 21 days, A39 and B10 localizations evolved as after phalloidin, but the changes were more marked. B1 was not affected by any of the treatments. In conclusion, colchicine, phalloidin and bile duct ligation do not seem to hinder the antigens in reaching the plasma membrane, but induce a redistribution of two of them, suggesting a disturbance in the biogenesis and/or control of the plasma membrane regionalization. Such an abnormal distribution could be involved in--or contribute to--the initiation of cholestasis.