Administration of combinations of manganese (Mn) and bilirubin (BR) to rats results in a severe, but reversible diminution of bile flow, an effect that can be abolished if sulfobromophthalein (BSP) is given at a specific time prior to BR. Some studies suggest that changes in the bile canalicular membrane (BCM) are critical to the response. One aim of the present work was to determine if functional changes in BCM also become more marked with increasing doses of BR. A second aim was to investigate the protective effects of BSP on MnBR-altered biliary function. The permeability of the biliary tree was evaluated by the segmented retrograde intrabiliary injection (SRII) procedure in male Sprague-Dawley rats treated with varying combinations and dosages of Mn, BR, and BSP. [3H]Mannitol and [3H]inulin were used as marker substances of the biliary tree (canalicular membrane and tight junctions, respectively). Administration of Mn, followed 15 min later by BR, led to a reduction in bile flow that was dose-dependent on BR. The percentage recovery of both inulin and mannitol in bile after SRII also decreased significantly with increasing dosages of BR. When BSP was given 10 min before BR, MnBR-induced reduction in bile flow was abolished. BSP treatment also prevented MnBR-induced reduction in biliary recovery of both inulin and mannitol after SRII; this was more evident with mannitol than with inulin. BSP protection against MnBR cholestasis depends upon when it is administered relative to BR injection. The relationship of BSP relative to BR injection was comparable for both reduced bile flow and the recoveries of marker substances in bile after SRII. The data are consistent with the conclusion that changes in biliary tree permeability, particularly at the canalicular membrane, likely lead to MnBR-induced cholestasis.