Ethanol administration inhibits the secretion of proteins by the liver, resulting in their hepatocellular retention. Experiments were designed in this study to determine the subcellular location of the retained secretory proteins. Ethanol was administered acutely to nonfasted rats by gastric intubation, whereas control animals received an isocaloric dose of glucose. Two hours after intubation, when maximum blood ethanol levels (45 mM) were observed, [3H]leucine and [14C]fucose were injected simultaneously into the dorsal vein of the penis. The labelling of secretory proteins was determined in the liver and plasma at various time periods after label injection. Ethanol treatment decreased the secretion of both leucine- and fucose-labeled proteins into the plasma. This inhibition of secretion was accompanied by a corresponding increase in the hepatic retention of both leucine- and fucose-labeled immunoprecipitable secretory proteins. At the time of maximum inhibition of secretion, leucine labeled secretory proteins located in the Golgi apparatus represented about 50% of the accumulated secretory proteins in the livers of the ethanol-treated rats, whereas the remainder was essentially equally divided among the rough and smooth endoplasmic reticulum and cytosol. Because fucose is incorporated into secretory proteins almost exclusively in the Golgi complex, fucose-labeled proteins accumulated in the livers of the ethanol-treated rats mainly in the Golgi apparatus, with the remainder located in the cytosol. These results show that ethanol administration causes an impaired movement of secretory proteins along the secretory pathway, and that secretory proteins accumulate mainly, but not exclusively, in the Golgi apparatus.