The contractile capacity of smooth muscle cells depends on the cytoskeletal framework of the cell. The aim of this study was to determine the functional importance of the microtubule, actin filament and intermediate filament components of the cytoskeleton in acetylcholine (ACh)-induced contractile responses of the rat isolated bronchial smooth muscle. The expressions of α-actin, β-actin, α-tubulin, desmin and vimentin were observed by immunoblotting in rat bronchial tissues. α-Actin and desmin were immunohistochemicaly observed in smooth muscle layer. Cytochalasin D, latrunculin A (inhibitors of the actin cytoskeleton) and acrylamide (an inhibitor of the intermediate filament) significantly decreased the contractions induced by ACh in concentration-dependent manners. On the other hand, colchicine or nocodazole (inhibitors of the microtubule cytoskeleton) had no effect on the ACh-induced contraction. These findings suggest that the contraction induced by ACh is highly dependent on polymerization of actin and intermediate filament, such as desmin, but not on the polymerization of microtubule in rat bronchial smooth muscle.