Several studies suggest that bile salts are transported from the basolateral to the canalicular membrane of hepatocytes by a vesicular pathway, possibly in part via the Golgi complex. To test this hypothesis, the present study examined, in the perfused rat liver, the influence of the Na+ionophore monensin on the biliary secretion of taurocholate and biliary lipids. The effects of the drug have been checked by the study of the ultrastructural modifications of the Golgi complex, secretion of horseradish peroxidase, and bile salt uptake. An infusion of monensin (1, 3, or 5 μmol/L) into the liver induced considerable swelling of the Golgi complex within 5 minutes. After a bolus injection of horseradish peroxidase during monensin infusion, the biliary secretion of the protein was delayed (1 μmol/L monensin) and markedly reduced (5 μmol/L monensin). Bile salt uptake was virtually unchanged except with 5 μmol/L monensin. This suggests that monensin has the same effects on the subcellular traffic in the perfused liver as in cultured cells. After a bolus injection of taurocholate (0.25, 5.0, or 8.5 μmol/100 g body wt) during monensin infusion, the pattern of biliary secretion of the bile salt was identical to that of controls. During continuous infusion of taurocholate, a 10-minute monensin infusion (1 or 3 μmol/L) had no effect on the biliary secretion of taurocholate and on the secretion of lecithin and cholesterol induced by taurocholate. High concentrations (5 μmol/L) or prolonged infusions (20 minutes) of monensin decreased the biliary secretion of bile salts but corresponded to a marked decrease of taurocholate uptake. In summary, the Na+ionophore monensin altered the Golgi complex and the vesicular transport of horseradish peroxidase, whereas taurocholate biliary secretion was not influenced unless taurocholate uptake by the liver was markedly decreased. It may be concluded that taurocholate and biliary lipid secretion, under these conditions, does not depend essentially on pathways involving acidic transporting vesicles and particularly the trans-Golgi complex.