Atherosclerosis is the most severe problem in the high-pressure systemic circulation and similar changes also occur in the high-pressure loading valve. This study was designed to test the hypothesis that early atherosclerosis, induced by a high cholesterol diet in rabbits, is characterized by significant ultrastructural change in the elastic laminae of the aortic valve. However, it is not known whether this process is also taking place in the cardiac valve at the early stage of atherosclerosis. Animals were fed either a high cholesterol diet (n = 5) or a control diet (n = 5) for 10-12 weeks. Histologic analysis demonstrated that subendothelial thickening and foam-cell infiltration were evident in the arterialis of aortic valves. Confocal microscopy revealed an altered pattern characterized by fragmentation and disorganization of the arterialis elastic laminae of hypercholesterolemic valves. Computerized digital analysis of the images obtained by confocal scanning microscopy demonstrated that compared to normal valves, the arterialis elastic laminae of hypercholesterolemic valves decreased in percentage of their elastin content (29.03 ± 1.10% vs. 42.94 ± 1.35%, p = 0.023). Immunohistochemical staining for matrix metalloproteinase-3 (MMP-3) revealed MMP-3 immunoreactivity was increased in hypercholesterolemic valves, predominantly in the arterialis. This study demonstrated that early atherosclerosis, induced by a high cholesterol diet in rabbits, is characterized by significant ultrastructural change in the elastic laminae of the aortic valve. The arterialis endothelium of the aortic valve may be a more atherosclerosis-prone area compared with the ventricularis. The presence of ultrastructural defect in the elastic laminae may play a role in chronic degenerative change and a resultant valvular dysfunction.