Estradiol-17-D-glucuronide (E₂17G) induces immediate, profound but transient cholestasis in rats when administered as a single bolus dose. Here we examined the consequence of sustained E₂17G cholestasis and assessed function and localization of the tight-junctional proteins zonnula occludens-1 (ZO-1) and occludin, and of the canalicular transporter Mrp2. An initial dose of E₂17G (15 µmol/kg, i.v.), followed by 5 subsequent doses of 7.5 µmol/kg from 60 to 240 min induced a sustained 40 - 70% decrease in bile flow. Following their biliary retrograde administration, cholera toxin B subunit-FITC or horseradish peroxidase were detected at the sinusoidal domain, indicating opening of the paracellular route; this occurred as early as 15 min after the first dose, as well as 15 min after the last dose of E₂17G, but not following administration of vehicle in controls. Localization of ZO-1 and occludin was only slightly affected under acute cholestatic conditions, but was severely disrupted under sustained cholestasis, with their appearance suggesting a fragmented structure. Endocytic internalization of Mrp2 to the pericanalicular region was apparent 20 min after single E₂17G-administration; however, Mrp2 was found more deeply internalized and partially redistributed to the basolateral membrane under sustained cholestasis. In conclusion, acute E₂17G-induced cholestasis increased permeability of the tight junction, while sustained cholestasis provoked a significant redistribution of ZO-1, occludin and Mrp2 in addition to increased permeability of the tight-junction. Altered tight junction integrity likely contributes to impaired bile secretion, and may be causally related to changes in Mrp2 localization.