Amylase secretion is induced by the accumulation of cAMP in response to beta-adrenergic stimulation and by the augmentation of intracellular Ca2+ in response to muscarinic-cholinergic stimulation in rat parotid glands. The roles of cytoskeleton and motor proteins in the secretory process are not yet known. We examined the effects of cytoskeleton-modulating reagents on the amylase release induced by isoproterenol (IPR) and carbamylcholine (Cch) in rat parotid acinar cells. The amylase release induced by Cch was decreased by the microtubule-disrupting reagent colchicine (Colch) and the myosin ATPase inhibitor 2,3-butanediene monoxime (BDM), but the release induced by IPR was not. The actin filament-stabilizing reagent jasplakinolide (Jasp) and actin filament-disrupting reagent cytochalasin D (CytoD) decreased the amylase release induced by both the beta-adrenergic and the muscarinic-cholinergic stimulants. Pretreatment with CytoD affected the shape of the acinar cells, which showed an intermediate state between the fusion of the secretory granules with the apical membrane and the retrieval of the membranes only after stimulation with IPR. Myosin and Dynein/dynactin complex were detected in the secretory granule membrane fraction. We concluded from this study that the cytoskeleton played different roles in the beta-adrenergic and the muscarinic-cholinergic secretory processes.