Acyl-CoA Cholesterol Acyltransferase (ACAT) (EC.2.3.1.26) catalyzes intracellular esterification of cholesterol with long chain fatty acyl-CoA. ACAT is an integral membrane protein of the endoplasmic reticulum membrane and plays a very important role in intracellular cholesterol homeostasis. Activity of ACAT is important for the production and secretion of lipoproteins and for intestinal absorption of dietary cholesterol. The involvement of ACAT activity in the pathogenesis of atherosclerosis and its related heart diseases leads to an extensive effort from both the pharmaceutical industry and the basic research community to study this protein and its catalytic mechanism. ACAT from yolk sac membranes of chicken eggs 16 days after fertilization has higher activity and better stability compared with the mammalian counterpart and plays a significant part in the supply of lipid nutrients as both structural components and energy source for developing embryos. ACAT of yolk sac membranes was studied in the current work and turned out to share many common features with its mammalian equivalent, including catalytically related histidine(s) and cysteine(s), detergent extractable activity, and possible cooperative interaction between cholesterol and ACAT. Differences in the properties of the enzyme exist between avian and mammalian sources and the most striking one is the lack of any effect of 25-hydroxycholesterol on the enzyme from yolk sac membrane. Redistribution of cholesterol within microsomal membranes accounts for the observed elevation of ACAT activity during storage at 4°C and factors in lipid bilayers may exist and restrict a complete random redistribution of cholesterol. The methyl ether of [beta]-cyclodextrin effectively extracts cholesterol from microsomal membranes upon repeated treatment, and it enhances cholesterol movement within microsomal fragments and between microsomal and liposomal vesicles, leading to enhanced activation of ACAT in microsomes and saturation of the activity when assayed with increasing concentration of cholesterol.