We examined the effect of ethanol administration on intravesicular pH in intact hepatocytes by applying a flow cytometric technique to detect fluorescein-isothiocyanate-dextran (FITC-dextran) in acidic vesicles. Rats were pair-fed liquid diets containing either ethanol or isocaloric carbohydrate for 1 to 5 weeks. Our study showed that ethanol administration increased the in situ pH of hepatic lysosomes by 0.15 to 0.2 pH units. This pH increase was sufficient to cause a significant reduction in lysosomal protein degradation. Long-term ethanol administration also caused a significant alkalinization of hepatic endosomes, and this increased pH was sustained over the course of vesicular acidification in hepatocytes incubated in vitro. Direct exposure of hepatocytes from rats fed control diet to either 25 mmol/L ethanol or 50 µmol/L colchicine also brought about a rapid alkalinization of acidic vesicles in a manner that resembled that seen in hepatocytes from ethanol-fed rats. These same treatments augmented the vesicular alkalinization already present in cells from ethanol-fed animals. Although ethanol administration had no effect on the content of the hepatic mannose-6-phosphate/IGFII receptor, the results indicate that sustained alkalinization of endosomes could have important functional consequences by impairing M-6-P/IGFII receptor recycling, thereby disrupting the delivery of newly synthesized hydrolases to lysosomes. This decreased complement of hydrolases within lysosomes together with alkalinization of the intralysosomal compartment would result in an overall decrease in lysosomal proteolysis.