Myocardial cells contain a cytoskeleton of intermediate filaments connecting the myofibrils. The present molecular analysis of the myocardial cytoskeleton was designed to identify the intermediate filament proteins and examine their assembly properties. The intermediate filament proteins desmin and vimentin were isolated from adult bovine myocardium by sequential extraction, urea solubilization, and chromatography on hydroxylapatite and DEAE columns. Desmin was obtained virtually pure in one peak and in a mixture of desmin and vimentin in the trailing fractions. Intermediate filaments of different morphologies polymerized in the desmin and the desmin-vimentin fractions. Isolated myocardial desmin occurs as three isozymes and isolated myocardial vimentin as two isozymes, which co-migrate on two-dimensional gels with corresponding isozymes from bovine skeletal and smooth muscle. Polypeptides of 200,000 and 220,000 daltons that fractionate with myocardial desmin and vimentin are also present in cytoskeletons of smooth and skeletal muscle. The results provide direct evidence that myocardial desmin can assemble to form intermediate filaments, suggesting that desmin is the major component of the cytoskeletal filaments in cardiomyocytes.