Cholesterol esterase of pancreatic juice origin and acyl coenzyme A:cholesterol acyltransferase, both associated with the intestinal mucosa, are implicated in the extensive esterification of exogenous cholesterol during absorption. To assess the role of each enzyme, [4-14C]cholesterol absorption into mesenteric lymph of rats with normal mucosal levels of both esterification enzymes was compared with that of rats with normal acyl coenzyme A:cholesterol acyltransferase activity but deficient cholesterol esterase activity. The cholesterol esterase deficiency was accomplished by either surgical diversion of the pancreatic juice from the intestinal lumen or removal by specific immunoprecipitation of cholesterol esterase from the otherwise complete pancreatic juice. In the rats that were transferase-complete and esterase-deficient, cholesterol absorption into lymph and esterase activity in the mucosa were decreased an average of 83% and 75%, respectively, compared with rats complete with both esterification enzymes. Of the absorbed [4-14C]cholesterol in all rats, 82-90% was esterified and the mucosal levels of cholesterol esterase, even in the esterase-deficient rats, could readily account for this esterification. Because transferase activity was normal in rat intestine in which cholesterol esterase was deficient and cholesterol absorption was inhibited, transferase alone does not support the absorption of exogenous cholesterol in the absence of esterase. These results reconfirm the importance of esterification in the absorption of exogenous cholesterol and demonstrate that cholesterol esterase plays an essential role in the regulation of the absorption process.