In order to demonstrate the effect to the acute administration of ethanol and chlorpromazine (CPZ) on bile flow and transhepatic transport of horseradish peroxidase (HRP) into bile, male rats were administered either 5 gm per kg ethanol intragastrically (E-rats) or 3 mg per kg CPZ intraperitoneally (CPZ rats). Control rats (C-rats) received saline. Two hours after ethanol feeding or 90 min after CPZ injection HRP was injected into the portal vein, and bile samples were collected at 1 0-min intervals for 2 hr. Tissue samples were removed at 1,10, 60, and 120 min to study HRP transport using electron microscopic cytochemical localization. Bile flow was reduced (p < 0.001) both in E- and CPZ-rats compared to C-rats. In E-rats HRP secretion was significantly decreased at 30 and 40 min post-HRP injection (p < 0.05) and the peak rate of HRP secretion was delayed by 10 min compared to C-rats. Uptake and transhepatic transport of HRP were similar to controls. These results suggest that bile secretion and flow were impaired by ethanol. CPZ inhibited secretion of HRP significantly (p < 0.001) during the first hr after HRP injection and by 25% after 2 hr. In CPZ rats studied cytochemically HRP reaction product decreased in the cytoplasm of hepatocytes 10 min after HRP injection (p < 0.01). These findings suggest that acute CPZ administration caused an inhibition of the uptake of HRP as well as secretion and bile flow.