The objective of the study was to characterize unesterified cholesterol particles in human aorta and to correlate the findings with the severity of aortic atherosclerosis. Human tissues were processed under conditions that preserve deposits of unesterified cholesterol agglomerates. Filipinfluorescence was determined by using a novel triple band pass filter. The pattern of unesterified cholesterol deposits was age related and correlated with the severity of atherosclerosis. We found three types of deposits: 1), small spherulites (3 to 5 mu), which were depicted in both the media and intima in individuals as early as age 16, and which, in more advanced ages, showed an increase in density and a tendency to aggregate extracellularly throughout the intima in clusters; 2), elongated structures (10 to 30 mu in the middle zone of the intima), the density of which was directly related to the severity of atherosclerosis; and 3), large (100 mu), irregular deposits found mainly in the core of atherosclerotic plaques. The medium size deposits, compared with those found in the core of atherosclerotic plaques, retain their overall size (10 to 30 mu), uniformity (oval elongated), and localization (middle zone of the intima). On the basis of these observations we hypothesize cholesterol deposition in two stages of aggregation: 1), early degradation of infiltrating low density lipoprotein particles forming unesterified cholesterol-rich vesicles in the vessel wall, followed by aggregation to spherulites in the lower part of the intima; and 2), more massive agglomeration of particles containing unesterified cholesterol and calcium phosphate in the midzone of the intima. Because in the second stage of aggregation the transition of cholesterol to the solid state has already occurred, it is irreversible.