Plasma high density lipoprotein (HDL) is inversely associated with the development of atherosclerosis. HDL exerts its atheroprotective role through involvement in reverse cholesterol transport in which HDL is loaded with cholesterol at the periphery and transports its lipid load back to the liver for disposal. In this pathway HDL is not completely dismantled but only transfers its lipids to the cell. Here we present evidence that a Chinese Hamster Ovarian cell line (CHO7) adapted to grow in lipoprotein deficient media degrades HDL and concomitantly internalizes HDL-derived cholesterol. Delivery of HDL cholesterol to the cell was demonstrated by a down-regulation of cholesterol biosynthesis, an increase in total cellular cholesterol content and by stimulation of cholesterol esterification after HDL treatment. This HDL degradation pathway is distinct from the low density receptor (LDL) receptor pathway, but also degrades LDL. 25-hydroxy cholesterol, a potent inhibitor of the LDL receptor pathway, down-regulated LDL degradation in CHO7 cells only in part, and did not down-regulate HDL degradation. Dextran sulfate released HDL bound to the cell surface of CHO7 cells and heparin treatment released protein(s) contributing to HDL degradation. The involvement of heparan sulfate proteoglycanes and lipases in this HDL degradation was further tested by two inhibitors genistein and tetrahydrolipstatin. Both blocked HDL degradation significantly. Thus, we demonstrate that CHO7 cells degrade HDL and LDL to supply themselves with cholesterol via a novel degradation pathway. Interestingly, HDL degradation with similar properties was also observed in a human placental cell line.