Smoothelin is a constituent of the cytoskeleton specific for smooth muscle cells (SMCs) in a broad range of species. It has been postulated that smoothelin represents a marker of highly differentiated, contractile SMCs. Here, we present data on the presence of smoothelin in the human vascular system that support this hypothesis. For this purpose, smoothelin distribution was studied (1) during vasculogenesis of the placenta, (2) in normal adult blood vessels, and (3) in atherosclerotic lesions. Smoothelin was first observed in placental tissue at approximately week 10 to 11 of gestation. In full-term placenta, it was found in the SMCs of vessels in the large stem villi and in the chorionic plate. Furthermore, it was present in the fetal arteries of smaller stem villi, but it was not found in the veins. In adult blood vessels, a small population of aortic (approximately 10%) and large muscular artery (approximately 30% to 50%) SMCs was positive for smoothelin. In general, smoothelin and desmin were coexpressed in the same SMCs, but expression of desmin appeared to be less abundant. However, the majority of SMCs in these blood vessels were smoothelin- and desmin negative but expressed vimentin, whereas alpha-smooth muscle actin (alpha-SMA) was present in all SMCs. The SMCs in the media of small muscular arteries were positive for smoothelin and desmin (> 95%), whereas the vimentin-positive SMC type was scarce. Smoothelin was absent in capillaries, pericytic venules, and small veins but was occasionally observed in the SMCs of large veins. Thus, the distribution of smoothelin in the SMCs of the vascular system appears to be limited to blood vessels that are capable of pulsatile contraction. In atherosclerotic femoral arteries, smoothelin-positive cells were detected in the media, the atheromatous plaque, and the intimal thickening. Smoothelin-positive cells were present primarily at the luminal portion of advanced lesions. The presence of a considerable number of such smoothelin-positive cells at that location may indicate that these plaques are no longer expanding.