Recently, we described a new pathway whereby peripheral cells can eliminate intracellular cholesterol by conversion into the more polar oxysterols 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid. The latter steroids are easily excreted from the cells and transported to the liver for conversion into bile acids. Our attempts to evaluate the importance of this new mechanism are reviewed here and also our investigations on the possible presence of additional similar pathways for removal of extrahepatic cholesterol. Human alveolar macrophages in culture were shown to have a high capacity to convert cholesterol into 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid and to excrete these steroids into the culture medium. Treatment of the macrophages with cyclosporin A, an inhibitor of sterol 27-hydroxylase, reduced the excretion of the 27-hydroxylated products by more than 90%, with a concomitant accumulation of intracellular cholesterol. The quantitative importance of the mechanism in relation to reverse cholesterol transport was investigated in 14C-cholesterol labelled macrophages exposed to HDL. At very low concentrations of HDL, possibly similar to those present in tissues, the two pathways were about equally effective. At optimal concentrations of HDL, however, reverse cholesterol transport was about 10-fold more effective than the sterol 27-hydroxylase pathway. The net uptake of 27-oxygenated steroids by the liver was measured in volunteers by comparison of the levels in the hepatic vein with those in a peripheral artery. Approximately 20 mg of 27-oxygenated oxysterols was taken up by the liver during 24 hours. Quantitative conversion of these oxysterols into bile acids would correspond to 4% of the total bile acid formation. It is evident that this new pathway contributes significantly to cholesterol elimination. The possibility that the sterol 27-hydroxylase pathway is of importance for cholesterol homeostasis in the brain was investigated by measuring oxysterols in the internal jugular vein and in an artery of healthy volunteers. There was no net flux of 27-hydroxycholesterol from the brain into the circulation. There was, however, a significant flux of 24-hydroxycholesterol, corresponding to elimination of about 4 mg cholesterol/24 hours. This flux is higher than the estimated rate of synthesis of cholesterol in the human brain. To summarize, we have demonstrated two mechanisms for cholesterol elimination from extrahepatic cells by specific oxygenases capable of oxidizing the steroid side-chain. The efficiency of these mechanisms is based on the fact that side-chain hydroxylated cholesterol species are both translocated through lipophilic membranes and converted into bile acids at a much faster rate than cholesterol itself. The importance of the sterol 27-hydroxylase-mediated mechanism is illustrated by the fact that patients who lack this enzyme develop xanthomas and premature atherosclerosis in spite of normal levels of circulating cholesterol.