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Over recent years, the role of matrix vesicles in the initial stages of arterial calcification has been recognized. Matrix calcifying vesicles have been isolated from atherosclerotic arteries and the biochemical composition of calcified vesicles has been studied. No studies have yet been carried out to examine the fine structure of matrix vesicles in order to visualize the features of the consequent stages of their calcification in arteries. In the present work, a high resolution ultrastructural analysis has been employed and the study revealed that matrix vesicles in human atherosclerotic lesions are heterogeneous with two main types which we classified. Type I calcified vesicles were presented by vesicles surrounded by two electron-dense layers and these vesicles were found to be resistant to the calcification process in atherosclerotic lesions in situ. Type II matrix vesicles were presented by vesicles surrounded by several electron-dense layers and these vesicles were found to represent calcifying vesicles in atherosclerotic lesions. To test the hypothesis that calcification of matrix vesicles surrounded by multilayer sheets may occur simply as a physicochemical process, independently from the cell regulation, we produced multilamellar liposomes and induced their calcification in vitro in a manner similar to that occurring in matrix vesicles in atherosclerotic lesions in situ.