Rats were given ethanol chronically (20-30% of the energy) in a nutritionally sufficient diet regimen. Controls received lipid as an isoenergetic substitute for ethanol. Protein synthesis in hepatocytes isolated from ethanol-fed rats was decreased compared with controls, but not in isolated nonparenchymal liver cells. Ethanol added in vitro inhibited protein synthesis in hepatocytes by 30%, but not in nonparenchymal cells for both ethanol-fed and control rats. Protein export and protein degradation in isolated hepatocytes were not affected by long-term ethanol treatment. Isolated hepatocytes were separated according to their buoyant density in linear metrizamide gradients. They were distributed in a bell-shaped manner regardless of donor rat treatment. Cells of low density contained three times as much lipid as high density cells. They were probably enriched in periportal cells, since histologic examination indicated a predominantly periportal localization of cells containing lipid droplets. Distribution of the intra-acinar marker alanine aminotransferase supported this conclusion. Protein synthesis was similar in the low-density hepatocyte populations of the respective groups of rats, whereas it was inhibited in a high-density population of ethanol-treated rats compared to the controls. Inhibition of protein synthesis by 80 mM ethanol was lower in the low-density hepatocytes of ethanol-fed rats.