Smooth muscle cells of the digestive, respiratory, and urogenital tracts contain desmin as their major, if not exclusive, intermediate-size filament constituent and also show a predominance of gamma-type smooth muscle actin. We have now examined smooth muscle tissue of different blood vessels (e.g., aorta, small arteries, arterioles, venules, and vena cava) from various mammals (man, cow, pig, rabbit, rat) by one- and two-dimensional gel electrophoresis of cell proteins and by immunofluorescence microscopy using antibodies to different intermediate-sized filament proteins. Intermediate-sized filaments of vascular smooth muscle cells contain abundant amounts of vimentin and little, if any, desmin. On gel electrophoresis, vascular smooth muscle vimentin appears as two isoelectric variants of apparent pI values of 5.30 and 5.29, shows the characteristic series of proteolytic fragments, and is one of the major cell proteins. Thus vimentin has been demonstrated in a smooth muscle cell present in the body. Vascular smooth muscle cells are also distinguished by the predominance of a smooth muscle-specific alpha-type actin, whereas gamma-type smooth muscle actin is present only as a minor component. It is proposed that the intermediate filament and actin composition of vascular smooth muscle cells reflects a differentiation pathway separate from that of other smooth muscle cells and may be related to special functions and pathological disorders of blood vessels.