Airway smooth muscle (ASM) cells in culture stiffen when exposed to contractile agonists. Such cell stiffening may reflect activation of the contractile apparatus as well as polymerization of cytoskeletal biopolymers. Here we have assessed the relative contribution of these mechanisms in cultured ASM cells stimulated with serotonin (5-hydroxytryptamine; 5-HT) in the presence or absence of drugs that inhibit either myosin-based contraction or polymerization of filamentous (F) actin. Magnetic twisting cytometry was used to measure cell stiffness, and associated changes in structural organization of actin cytoskeleton were evaluated by confocal microscopy. We found that 5-HT increased cell stiffness in a dose-dependent fashion and also elicited rapid formation of F-actin as marked by increased intensity of FITC-phalloidin staining in these cells. A calmodulin antagonist (W-7), a myosin light chain kinase inhibitor (ML-7) and a myosin ATPase inhibitor (BDM) each ablated the stiffening response but not the F-actin polymerization induced by 5-HT. Agents that inhibited the formation of F-actin (cytochalasin D, latrunculin A, C3 exoenzyme, and Y-27632) attenuated both baseline stiffness and the extent of cell stiffening in response to 5-HT. Together, these data suggest that agonist-evoked stiffening of cultured ASM cells requires actin polymerization as well as myosin activation and that neither actin polymerization nor myosin activation by itself is sufficient to account for the cell stiffening response.