The distribution of tubulin and desmin, the constituent proteins of microtubules and intermediate filaments, respectively, were studied in normal and hypertrophied rat myocardium by high-resolution immunofluorescence and immunoelectron microscopy. Cardiac hypertrophy was induced in 25-day-old rats by aortic stenosis. In the normal heart, double immunolabelling of ultrathin frozen sections of papillary muscle using gold-labelled probes for tubulin and desmin showed that microtubules ran primarily in a longitudinal direction through the intermyofibrillar spaces, perpendicularly to the desmin filaments. Microtubules were present near nuclei, mitochondria, and plasma membranes, while desmin filaments formed transverse connections between adjacent Z disks. No tubulin was observed near the intercalated disks, which were rich in desmin filaments. In hypertrophied hearts, myocytes exhibited the typical morphological features of developing hypertrophy. While there was little difference in the distribution of the microtubules around mitochondria and at the plasma membrane, considerable increases were seen near the nuclei and along the myofibrils. Desmin labelling was distributed transversely as in the controls; however, sometimes it was longitudinally oriented either in the intermyofibrillar space linking 2 Z disks out of register or along digitations of the intercalated disks connecting neighboring desmosomes. The unique rearrangement of desmin and tubulin filaments in hypertrophied cardiac myocytes emphasizes their distinct role in myocyte organization. We suggest that, during the development of cardiac hypertrophy, desmin filaments are mainly involved in maintaining the myofibrils in register, whereas the degree of assembly of microtubules is correlated with the rate of protein synthesis and with myofibrillogenesis.