Bile salt stimulated cholesterol esterase is predominantly synthesized in the pancreas. However, this enzyme is also synthesized by the liver and was found to be present in plasma. The physiologic role of the systemic cholesterol esterase has not been clearly defined. In the current study, the human hepatoma cell line HepG2 was used as a model to determine the role of cholesterol esterase on hepatic uptake of high density lipoprotein (HDL)-associated cholesteryl esters. The results showed that hepatic uptake of the cholesteryl esters analog [3H]cholesteryl ether on reconstituted HDL was inhibited by anti-cholesterol esterase antibodies. The HDL-associated cholesteryl ester transported to HepG2 cells was also increased 2-fold in the presence of taurocholate, an activator of the cholesterol esterase. These results suggest that liver-derived cholesterol esterase may play an important role in cellular uptake of cholesteryl esters from HDL. This hypothesis was supported by demonstrating the ability of exogenously added cholesterol esterase to further enhance hepatic uptake of HDL-associated cholesteryl esters. The results of the current study also showed that cholesterol esterase increased free-to-esterified cholesterol ratio in the lipoprotein. Thus, alteration of HDL structure and composition contributes to the cholesterol esterase-induced cellular uptake of HDL-associated cholesteryl esters. On the basis of these observations, we propose that liver-derived cholesterol esterase may play an important role in lipoprotein metabolism.