Although disruption of the microtubule (MT) array inhibits myogenesis in myocytes,.the relationship between the assembly of microtubules (NIT) and the organization of the contractile filaments is not clearly defined. We now report that the assembly of mature myofibrils in hypertrophic cardiac myocytes is disrupted by myoseverin, a compound previously shown to perturb the NIT array in skeletal muscle cells. Myoseverin treated cardiac myocytes showed disruptions of the striated Z-bands containing alpha-actinin and desmin and the localization of tropomyosin, titin and myosin on mature sarcomeric filaments. In contrast, NIT depolymerization by nocodazole did not perturb sarcomeric filaments. Similarly, expression of constitutively active stathmin as a non-chemical molecular method of NIT depolymerization did not prevent sarcomere assembly. The extent of NIT destabilization by myoseverin and nocodazole were comparable. Thus, the effect of myoseverin on sarcomere assembly was independent of its capacity for NIT inhibition. Furthermore, we found that upon removal of myoseverin, sarcomeres reformed in the absence of an intact MT network. Sarcomere formation in cardiac myocytes therefore, does not appear to require an intact NIT network and thus we conclude that a functional NIT array appears to be dispensable for myofibrillogenesis.