The fluorescent probe, filipin, and the lipid-soluble dye, oil red O, have been used to simultaneously detect and differentiate free and esterified cholesterol, respectively, in tissue sections prepared from spontaneous atherosclerotic lesions of swine. This was possible because filipin stains free cholesterol but does not stain cholesteryl ester and because oil red O stains cholesteryl ester but does not stain free cholesterol. Oil red O-stained lipid accumulated intra- and extracellularly but separate from filipin-stained lipid. Spherical filipin-stained particles and elongated filipin-stained crystals accumulated in the extracellular space. Interestingly, some cells appeared to accumulate these filipin-stained particles exclusively. The spherical filipin-stained particles have not been previously recognized because they are not stained by oil red O. This and the fact that extensive compartmentalization of filipin and oil red O-stained lipid occurs in atherosclerotic lesions are new observations to be considered in the pathogenesis of vascular cholesterol accumulation.