Administration of phalloidin, one of the toxic peptides of the mushroom Amanita phalloides, leads to rapid and sustained cholestasis in rats. Although attributed to the interaction of phalloidin with microfilaments, the events leading to cholestasis are incompletely understood. The adenosine triphosphate (ATP)-dependent, apical conjugate export pump, termed multidrug resistance protein 2 (Mrp2) or canalicular multispecific organic anion transporter, is the major driving force for bile salt-independent bile flow. We investigated the role of Mrp2 in phalloidin-induced cholestasis. Bile flow decreased to 53% and 31% of control at 15 and 30 minutes after phalloidin (0.5 mg/kg), respectively. Mrp2-mediated [3H]leukotriene excretion into bile during the initial 45 minutes was reduced to 44% of control when [3H]LTC4 was injected 15 minutes after phalloidin treatment. Mrp2 was progressively lost from the hepatocyte canalicular membrane and detected predominantly on intracellular membrane structures together with other canalicular proteins including P-glycoproteins, ecto-ATPase, and dipeptidyl-peptidase IV. By contrast, structures involved in intercellular adhesion (zonula occludens, zonula adhaerens, and desmosomes) as well as intermediate filaments of the cytokeratin type appeared largely unaffected within 30 minutes after phalloidin. In line with the immunofluorescence analysis, immunoblots indicated a loss of Mrp2 and P-glycoproteins from the canalicular membrane and a 3- and 4.6-fold increase of these transport proteins in the microsomal fraction, respectively. Our results indicate that phalloidin induces marked alterations of the hepatocyte canalicular architecture and a loss of Mrp2 together with other proteins from the canalicular membrane. The resulting cholestasis can therefore be explained in part by the loss of export pumps, including Mrp2, from the canalicular membrane.