The effect of an extract of the toxic mushroom Amanita phalloides (Fr. Secr.) on the transfer of metabolically inert, water-soluble solutes across the biliary tree epithelium of male Sprague-Dawley rats was studied by the segmented retrograde intrabiliary injection (SRII) technique. The purpose was to see if indications for a change in the permeability of the biliary tree could be obtained in vivo. This technique involved the introduction into the biliary system, through a bile duct cannula, of an initial “segment” of solution containing a radioactive marker compound followed by a volume of 0.9% saline given in excess of the biliary tree capacity. Bile flow was immediately reestablished and bile drops were serially collected. The radioactive content was determined and expressed as a percentage of the total administered dose of radioactivity. Evaluated 24 hr following the ip administration of an extract of Amanita phalloides, the biliary recoveries of [3H]mannitol, [3H]sucrose, [3H]inulin, and [3H]dextran were all found to be significantly decreased when compared to controls. These decreased recoveries were consistent with an increase in hepatobiliary permeability. This increase in permeability was not accompanied by any change in liver weight or bile flow. Bile duct ligation, 24 hr before administration of a lethal dose of the extract, protected rats against the alteration in permeability as well as the lethality. Bile duct ligation itself increased bile flow and increased the distended biliary tree capacity.