The movement of cholesterol from high-density lipoproteins (HDL) into bile has been studied using perfused livers from cholesterol-fed African Green monkeys. Mass amounts of HDL were isolated from the plasma of African Green monkeys and were doubly labeled with either 125I-apolipoprotein and [3H]cholesteryl ester or with [3H]cholesteryl ester and [14C]cholesterol. For 3 h of perfusion HDL-free cholesterol was cleared from perfusate at a faster rate than HDL ester cholesterol which, in turn, was cleared at a faster rate than HDL protein. [14C]cholesterol from HDL appeared in biliary bile acids and cholesterol at a higher rate than [3H]esterified cholesterol from HDL. The specific activities of biliary [14C]cholesterol and HDL-free [14C]cholesterol had equilibrated by 60 min of perfusion, although the specific activity of whole liver free [14C]cholesterol was still only 46% of that in bile at 180 min of perfusion. In contrast, the specific activity of total liver free [3H]cholesterol was equal to that of biliary [3H]cholesterol by 180 min of perfusion. We conclude that, in this primate model, HDL-free cholesterol enters into a hepatic compartment that communicates with biliary cholesterol and bile acid precursor pools more efficiently than with other liver pools of cholesterol, whereas HDL-esterified cholesterol appears to mix with all liver pools with equal efficiency. Overall, these data support the concept of compartmentalization of cholesterol in the liver.