The output of lipids and lipoproteins by isolated perfused livers of normal-fed and cholesterol-fed rabbits has been examined. There was a comparable output of triglyceride by the livers of both groups, resulting in an accumulation of 40-50 mg triglyceride/liver/2 h in the perfusate in each case. The output of cholesteryl esters, however, was very much greater from the livers of cholesterol-fed (45 mg/liver/2 h) than from normal-fed (3.3 mg/liver/2 h) rabbits. The major lipoproteins in liver perfusates from both groups of animals were very low density lipoproteins (VLDL). In the perfusate of normal livers the VLDL were enriched with triglyceride and depleted of cholesteryl esters when compared with plasma VLDL from normal animals. VLDL in the perfusate of livers from cholesterol-fed rabbits, on the other hand, were markedly enriched with cholesteryl esters; cholesteryl esters accounted for 33% by mass of VLDL from cholesterol-fed livers and only 3.1% of VLDL from normal livers. The cholesteryl esters in the plasma lipoproteins of cholesterol-fed rabbits were relatively enriched with cholesteryl oleate when compared to those in normal plasma. Similarly, cholesteryl oleate predominated in the VLDL in the liver perfusate of the cholesterol-fed animals, consistent with an hepatic acyl CoA/cholesterol acyltransferase origin. Thus, cholesterol-feeding in the rabbit results in a marked increase in the hepatic secretion of cholesteryl esters as a component of VLDL.