Bile salt-stimulated lipase (BSSL), or carboxyl ester lipase, is a constituent of exocrine pancreatic secretion and, in some species, including humans, also of milk. BSSL has been suggested to have a direct effect on intestinal uptake of dietary cholesterol besides being the key enzyme in the hydrolysis of fat-soluble vitamins and cholesterol esters. Furthermore, an intestinal heparin-containing receptor for the enzyme has been implicated. If BSSL promotes dietary cholesterol utilization, this might be of particular importance in the neonatal period, which is characterized by a high need of cholesterol for membrane synthesis. We have studied binding of BSSL to intestinal membranes in vitro and if such binding affects the uptake of cholesterol. BSSL bound avidly to rat intestinal microvesicle membranes and the binding was inhibited by addition of free heparin or heparin fragments. In this model system, we could not demonstrate any effect of BSSL on cellular uptake of free cholesterol. However, if esterified rather than free cholesterol was present in the incubation, hydrolysis by BSSL was the rate-limiting step in cellular cholesterol uptake. We therefore conclude that BSSL is important for utilization of dietary cholesterol only by hydrolyzing cholesterol esters and not by acting as a transport protein.