Cultured airway smooth muscle cells subjected to cyclic strain respond with increased cytoskeletal organization and contractility resembling effects described with RhoA activation. To test the hypothesis that strain increases cell cytoskeletal organization through RhoA, cells were subjected to strain in the presence of known activators or inhibitors of RhoA. Ten percent cyclic deformational strain (serum-free conditions) increased F-actin staining (152% over control), and this effect was enhanced by serum or lysophosphatidic acid (180%), but decreased (68%) with Clostridium botulinum toxin inhibition of RhoA or with the Rho kinase inhibitor Y27632 (67%). When cells expressing the dominant negative N17-RhoA isoform were subjected to strain, F-actin staining was disorganized and cells failed to elongate or migrate relative to strain direction. When cells expressing a green fluorescent protein (GFP)-RhoA fusion protein were subjected to strain, GFP showed up to 25% greater cell membrane staining than control cells. Finally, strain caused a 4-fold increase in RhoA activation (Rhotekin binding assay), and a 3-fold increase myosin phosphatase phosphorylation that was inhibited by Y27632. We conclude that mechanical stress activates RhoA, an event that may increase airway smooth muscle contractility.