—This study was designed to identify cellular responses associated with free cholesterol (FC) accumulation in model macrophage foam cells. Mouse peritoneal macrophages (MPMs) or J774 macrophages were loaded with cholesteryl esters using acetylated LDL and FC/phospholipid dispersions and were subsequently exposed to an acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor. This treatment produced a rapid accumulation of cellular FC. The FC that accumulated due to ACAT inhibition was more readily available for efflux to 2-hydroxypropyl-β-cyclodextrin (which removes cholesterol from the plasma membrane) than FC in untreated control cells. After a 3-hour exposure to an ACAT inhibitor, a significant increase in phospholipid synthesis was seen, followed by the leakage of LDH after 12 hours of treatment. We also observed, by electron and fluorescence microscopy, morphological indications of both apoptosis and necrosis in cells treated with an ACAT inhibitor. In addition, inhibition of ACAT for 48 hours resulted in the formation of FC crystals in MPMs but not in J774 cells. If compound 3β-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A), which modulates intracellular trafficking of cholesterol, was added together with the ACAT inhibitor, each of the metabolic changes elicited by the accumulation of excess FC was either diminished or eliminated. The protective affect of U18666A was not due to a decrease in cellular FC concentrations, because cells treated with an ACAT inhibitor accumulated similar amounts of FC in the presence or absence of U18666A. Thus, treatment with U18666A results in the sequestering of FC in a pool that prevents it from causing various responses to FC deposition in macrophages. The metabolic changes that were produced when these model foam cells were treated with the ACAT inhibitor parallel the pathological events that have been shown to occur in the developing atherosclerotic plaque.