Smoothelin is a constituent of the smooth muscle cell (SMC) cytoskeleton. Antibodies directed to smoothelin comprise a useful tool to monitor SMC differentiation. Smoothelin is exclusively expressed in fully differentiated (contractile) SMCs. RNA and protein analyses revealed a broad species distribution of this protein. Cells with SMC-like characteristics, such as myofibroblasts and myoepithelial cells as well as skeletal and cardiac muscle, do not contain smoothelin. Confocal scanning laser microscopy of tissue sections show a filamentous organization of smoothelin, colocalizing with actin stress fibers. In immunoblots two molecular weight isoforms are detected, i.e., a 59kD isoform specific for visceral SMC (Smoothelin A) and an isoform with a molecular weight of ~100kD in vascular SMC (Smoothelin B). Human smoothelin is encoded by a single copy gene which is located on chromosome 22. The monoclonal smoothelin antibody is useful for discriminating fully differentiated, contractile SMCs from proliferative SMCs. The distribution of smoothelin-positive cells indicates a correlation between smoothelin expression and smooth muscle tissue contractility. Smoothelin is a useful tool in the evaluation of atherosclerotic lesions.