Due to its presumed role in regulating cellular cholesterol homeostasis, and in various pathophysiological conditions, acyl-coenzyme A:cholesterol acyltransferase (ACAT) has attracted much attention. Cloning the ACAT gene provides the necessary tool to advance molecular studies of this enzyme. The topics reviewed in this chapter include the pathophysiological roles of ACAT, the biochemistry and molecular biology of the ACAT protein and the ACAT gene, and the mode of regulation by sterol or nonsterol agents in mammalian cells. In addition, we present a working model linking the presumed allosteric property of ACAT with cholesterol trafficking into and out of the endoplasmic reticulum.