Cholesteryl esters, formed by the esterification of cholesterol with long-chain fatty acids, on one hand, are the means by which cholesterol is transported through the blood by lipoproteins, on the other, the way cholesterol itself can be accumulated in the cells. Therefore, these important molecules play an active part in metabolic pathways that form the basis of cholesterol trafficking and homeostasis. The role of different regulatory mechanisms in cholesterol homeostasis in physiologic and neoplastic conditions with emphasis on intracellular content of cholesteryl esters is here reviewed. Numerous studies carried out on tumor cell lines, experimental tumors, and human tumors have shown an abnormal cholesterol metabolism that is reflected by an increase in intracellular cholesteryl esters due to an alteration in all the mechanisms that form the basis of regulation, in particular: cholesterol de novo biosynthesis; uptake of exogenous cholesterol LDL receptor mediated; cholesterol esterification mediated by the ACAT activity; cholesterol efflux HDL receptor mediated. The most recent analytic-spectroscopic applications that permit cholesteryl ester determination on tumor lipidic extracts and directly in vivo are also reported. This review gives an overview of cholesterol homeostasis in physiological and pathological conditions where cholesteryl esters are over-expressed.