Atherosclerotic calcification may weaken the aorta wall and thereby lead to rupture of the vessel. The mechanism whereby aortas undergo calcification remains unclear. Previous reports in this laboratory showed that, after 2 months of cholesterol-supplemental feeding, an increase in calcifiability of membrane vesicles isolated from rabbit aortas precedes substantial arterial calcification. Further, the mineral was deposited by isolated calcifiable vesicles as an amorphous phase similar to minerals in human aortas at an early stage of atherosclerosis. In the current study, atherosclerotic calcification was induced by exposing rabbits to a 1% cholesterol-rich diet for 3 or 6 months. After 3 months of dietary interventions, atherosclerotic lesions were fully developed. Fatty streaks were evident in areas proximal to the heart and became less frequent in the distal areas. However, calcification was not yet identifiable histologically or by using Fourier transform spectroscopy (FT-IR). After 6 months of high cholesterol treatment, aortas were partially calcified. Histochemical staining for mineral revealed that calcification appeared to occur predominantly in the intimal areas immediately adjacent to the media. Fourier Transform Imaging analysis demonstrated that the mineral deposited in atherosclerotic rabbit aortas was a hydroxyapatite-like phase. To determine whether aorta vesicles play a role in mineral formation in aortas, vesicles were isolated from calcified aortas and then their calcifiability was compared to that in normal vesicles. Interestingly, during the course of vesicle isolation, we found that calcifiable vesicles with much higher calcifiability than normal vesicles could be readily isolated from atherosclerotic aortas simply by suspending minced tissues in PBS. The characteristics of the calcification process and the enzymatic contents of isolated vesicles were similar to those obtained using collagenase digestion. Correlatively, mineral deposited by calcifiable vesicles isolated from the calcified aortas was also of hydroxyapatite-like phases. Altogether, these observations indicate that (1) aortic calcification is a later event during atherogenesis, (2) calcifiable vesicles are loosely bound to the matrices of the lesions as the result of the disease process and (3) similarities in the mineral phases between those in aortas and by vesicles during atherogenesis further support the role of calcifiable vesicles in dystrophic calcification.