Pancreatic secretion is required for efficient cholesterol absorption by the intestine, but the factors responsible for this effect have not been clearly defined. To identify factors involved and to investigate their role in cholesterol uptake, we studied the effect of Viokase(R), a porcine pancreatic extract, on cholesterol uptake into human intestinal Caco-2 cells. Viokase is capable of facilitating cholesterol uptake into these cells such that the level of uptake is 5-fold higher in the presence of solubilized Viokase. This stimulation is time-dependent and is dependent on the presence of bile salt. However, bile salt-stimulated pancreatic cholesterol esterase, which has been proposed to mediate cholesterol uptake, is not fully responsible. The major cholesterol transport activity was purified and identified as pancreatic phospholipase A2. Anti-phospholipase A2 antibodies abolished virtually all of the phospholipase A2 and cholesterol transport activity of solubilized Viokase. We demonstrate that both phospholipase A2 and cholesterol esterase increase cholesterol uptake by hydrolyzing the phosphatidylcholine that is used to prepare the cholesterol-containing micelles. In the absence of cholesterol esterase or phospholipase A2, uptake of cholesterol from micelles containing phosphatidylcholine is not as efficient as uptake from micelles containing phospholipase A2-hydrolytic products. These results indicate that phospholipase A2 may mediate cholesterol absorption by altering the physical-chemical state of cholesterol within the intestine.