While recent data in intrahepatic bile duct epithelial cells (IBDECs) isolated from normal rat liver have established their ability to undergo endocytosis, few studies have assessed endocytosis in IBDECs in situ. Thus, to clarify the activity of IBDECs in situ on macro-molecules in bile and blood, we injected horseradish peroxidase (HRP) into either the common bile duct or the portal vein, and determined its intracellular distribution by electron microscopic cytochemistry. Successful retrograde injection into the common bile duct was achieved by resection of the liver surface so as to avoid HRP leakage from the bile duct on injection. IBDECs internalized HRP through both the apical and basolateral plasma membranes. By quantitative analysis, counting the number of HRP-positive vesicles in the cells, apical endocytosis was found more active than basolateral. HRP internalized through the apical membrane was either routed to the acid phosphatase-positive lysosomes for degradation or to extracellular space for transcytosis. HRP through the basolateral membrane was transferred to the organelles having lipid inclusion, which were expected to be lysosomes negative for acid phosphatase. Our results suggest that IBDECs in situ are actively engaged in endocytosis for degradation of macromolecules in bile and blood, and possibly engaged in the excretion of macromolecules into extracellular space.