Mutant rats (TM rats) with abnormal hepatic excretory function were used to study biliary transport of dibromosulfophthalein, ouabain, tributylmethyl ammonium, cholate and taurocholate. In whole animals, dibromosulfophthalein and ouabain clearance is reduced to 7 and 37% of normal, respectively, due to severely impaired excretion from liver to bile. Initial uptake rates of these agents are relatively little affected. In the isolated perfused liver preparation, dibromosulfophthalein is retained within liver and perfusion medium, and the 60-min recovery on bile is reduced to 1.5 vs 75% in normal controls. Biliary excretion of cholate, taurocholate and the quaternary ammonium cation, [14C]tributylmethyl ammonium, is not impaired. These results provide evidence for a selective defect of organic anion and neutral steroid transport in TM rats and confirm that multiple pathways exist for the hepatobiliary excretion of organic anions, neutral steroids, bile acids and cations. Bile flow in whole animals and in the isolated perfused liver is reduced to 50 and 30% of normal, respectively. This suggests that a normal function of the excretory systems for organic anions and neutral steroids is important for the maintenance of normal bile flow.