Cholesterol monohydrate crystals are frequently detected in intermediate and advanced atherosclerotic lesions. Little is known regarding mobilization of this molecular form of cholesterol into metabolically active pools. To study a potential mechanism for mobilization of crystalline cholesterol, we examined its uptake by a mouse macrophage cell line (P388D1). Crystals were overlayered on a P388D1 cell monolayer maintained in a serum-free medium. Following incubation, the monolayer was washed, and the cells were harvested and analyzed for crystal internalization. By transmission electron microscopy, crystals were found intracellularly surrounded by a bilayer membrane. Analyses of the cellular cholesterol ester content by gas-liquid chromatography and esterification of [14C]cholesterol indicated the conversion of crystalline cholesterol to cholesterol esters. This pathway for solubilization of cholesterol crystals by macrophages could play an important role in the regression of atherosclerotic lesions.