The microsomal enzyme acyl-CoA:cholesterol acyltransferase (ACAT; EC 2.3.1.26) catalyzes the esterification of cellular cholesterol with fatty acids to form cholesterol esters. ACAT activity is found in many tissues, including macrophages, the adrenal glands, and the liver. In macrophages, ACAT is thought to participate in foam cell formation and thereby to contribute to atherosclerotic lesion development. Disruption of the gene for ACAT (Acact) in mice resulted in decreased cholesterol esterification in ACAT-deficient fibroblasts and adrenal membranes, and markedly reduced cholesterol ester levels in adrenal glands and peritoneal macrophages; the latter finding will be useful in testing the role of ACAT and macrophage foam cell formation in atherosclerosis. In contrast, the livers of ACAT-deficient mice contained substantial amounts of cholesterol esters and exhibited no reduction in cholesterol esterification activity. These tissue-specific reductions in cholesterol esterification provide evidence that in mammals this process involves more than one form of esterification enzyme.