Actin filaments align around myosin filaments in the correct polarity and in a hexagonal arrangement to form cross-striated structures. It has been postulated that this myosinactin interaction is important in the initial phase of myofibrillogenesis. It was previously demonstrated that an inhibitor of actin-myosin interaction, BDM (2,3-butanedione monoxime), suppresses myofibril formation in muscle cells in culture. However, further study showed that BDM also exerts several additional effects on living cells. In this study, we further examined the role of actin-myosin interaction in myofibril assembly in primary cultures of chick embryonic skeletal muscle by applying a more specific inhibitor, BTS (N-benzyl-p-toluene sulphonamide), of myosin ATPase and actin-myosin interaction. The assembly of sarcomeric structures from myofibrillar proteins was examined by immunocytochemical methods with the application of BTS to myotubes just after fusion. Addition of BTS (10–50 μM) significantly suppressed the organization of actin and myosin into cross-striated structures. BTS also interfered in the organization of α-actinin, C-protein (or MyBP-C), and connectin (or titin) into ordered striated structures, though the sensitivity was less. Moreover, when myotubes cultured in the presence of BTS were transferred to a control medium, sarcomeric structures were formed in 2–3 days, indicating that the inhibitory effect of BTS on myotubes is reversible. These results show that actin-myosin interaction plays a critical role in the process of myofibrillogenesis.