Only a few previous works investigated the involvement of Chlamydia pneumoniae (Chlamydophila pneumoniae) in arterial calcification. The present study investigated a possible association between C. pneumoniae and medial calcification. Carotid artery segments obtained by endarterectomy from 60 patients were examined by polymerase chain reaction (PCR) and immunohistochemistry to identify the presence of C. pneumoniae. Arterial specimens showing double positive (n=17), double negative (n=22) and single positive results (n=21) were further analysed by a combination of histology, immunohistochemistry and electron microscopy. Medial calcification occurred in 10 of 17 C. pneumoniae double positive arterial specimens (58.8%) but no medial calcification was observed in any of 22 C. pneumoniae double negative arterial specimens. Electron microscopy indicated the presence of C. pneumoniae in smooth muscle cells in foci of medial calcification. Medial smooth muscle cells showing damage of the cytoplasm and basement membrane contained the structures with appearance of elementary, reticulate and aberrant bodies of C. pneumoniae. The presence of C. pneumoniae in smooth muscle cells was confirmed by electron-microscopic immunocytochemistry. In the extracellular matrix, C. pneumoniae aberrant bodies exited from smooth muscle cells were observed to undergo calcification. The findings offer a new hypothesis of arterial calcification. The findings suggest that the infection of medial smooth muscle cells with C. pneumoniae may be associated with the pathophysiological events of arteriosclerotic calcification of the tunica media.