Diethylmaleate is used as a model compound whose glutathione conjugates are secreted into bile, and which induce choleresis and the formation of Golgi-derived vesicles in hepatocytes. This study was performed to test the assumption that these vesicles are involved in the bile canalicular secretion of diethylmaleate. We reasoned that phalloidin and colchicine, two drugs acting on microfilaments and microtubules, respectively, can modify the movements of diethylmaleate-induced vesicles towards the bile canaliculus. Phalloidin induced the formation of a thick microfilamentous network around the bile canalicular plasma membrane domain. A significant decrease in diethylmaleate-stimulated choleresis was observed, associated with a striking accumulation of pericanalicular vesicles, which were confirmed by morphometric analysis. In contrast, in rats pretreated with colchicine, after diethylmaleate administration, only a few vesicles were observed around the bile canaliculus, while diethylmaleate-induced choleresis also decreased. These results suggest that: a) the thick microfilament network induced by phalloidin prevents diethylmaleate-associated vesicles reaching the bile canalicular plasma membrane; and b) colchicine produces a dispersion of these vesicles in the cytoplasm of hepatocytes by inhibiting the polymerization of microtubules. These observations support a role of vesicles in the transport of diethylmaleate by hepatocyte into bile, and are consistent with the existence of a vesicular pathway for the biliary secretion of diethylmaleate and possibly other organic anions.