In our previous report, we have described morphological changes in hepatocytes, e.g., enlargement of mitochondria and a change in the lamellar formation of rough endoplasmic reticulum, produced by short-term feeding of a protein-free diet to rats. In order to examine whether or not these morphological changes in the subcellular organella are accompanied by any functional and compositional changes, we measured the P/O ratio and respiratory rate of mitochondria, and the phospholipid fatty acids of the mitochondrial and microsomal membranes in the liver of rats fed a protein-free diet for a short period. Feeding rats the protein-free diet for 4 days or 27-28 days had no effect on the rate of hepatic mitochondrial oxygen consumption (State 3 respiration). The diet significantly decreased the P/O ratio on the 4th day, but did not affect it on the 27-28th days. The decreased P/O ratio observed on the 4th day returned to the control level after overnight refeeding of a 20% casein diet. Main compositional changes induced by feeding rats the protein-free diet for 2 days were significant decreases in the phospholipid/protein ratios of the total liver and mitochondrial inner membrane, a tendency of an increase in the ratio of phosphatidylcholine (PC) to total phospholipids in the mitochondrial outer membrane, a significant decrease and a tendency of decrease in the arachidonate/linoleate ratio in the phosphatidylethanolamine (PE) and PC, respectively, in the mitochondrial outer membrane. Some of these results were discussed in relation to the morphological changes in mitochondria and rough endoplasmic reticulum produced by short-term feeding of the protein-free diet which we previously reported.