1. The synthesis of glycerolipids from a number of radioactive precursors, such as [2-3H] glycerol, [32P]phosphate, [Me-14C]choline and [1,2-14C2]ethanolamine proceeds in enzymatically isolated hepatocytes with a specificity that agrees very well with that observed in the liver in vivo. 2. The nutritional state of the rat has a profound influence on the glycerolipid metabolism of isolated hepatocytes. Fasting strongly decreased the incorporation of glycerol via sn-glycerol 3-phosphate into triacylglycerols whereas the formation of phosphatidylcholine and, particularly, phosphatidylethanolamine was much less affected by food deprivation. Refeeding a high-sucrose, fat-free diet caused a tremendous increase in the synthesis of diacylglycerols and triacylglycerols to values exceeding those found in hepatocytes from normally fed animals. The formation of phosphatidylcholine increased more slowly and the synthesis of phosphatidylethanolamine was even depressed by refeeding a high-sucrose, fat-free diet. 3. Alteration of the nutritional state resulted in similar changes in the amounts and metabolism of hepatic glycerolipids in vivo. The formation and, consequently, the amount of diacyglycerol and triacylglycerol were decreased by fasting and increased to values above normal in rats refed a high-sucrose, fat-free diet. The formation and the amounts of phospholipids in the liver decreased slightly by fasting which was mainly due to a decrease in phosphatidylcholine. Refeeding caused a significant increase in the formation and amount of phosphatidylcholine. The amount of phosphatidylethanolamine was even further diminished by feeding a high-sucrose, fat-free diet to 48-h-fasted rats. 4. These results show that the alterations, induced in the in vivo metabolism of hepatic glycerolipids, by changes of the dietary state, are also reflected in the isolated hepatocytes. This finding strengthens the potential significance of isolated hepatocytes in studies on the regulation of hepatic lipid metabolism.