Abnormal mechanical stress on pulmonary structures is associated with increased airway resistance and impaired gas exchange as a result of increased airway smooth muscle (ASM) deposition. Using an in vitro system with cultured ASM cells, we have demonstrated that cyclic deformational strain increases ASM cellular myosin and myosin light chain kinase. To determine if these contractile protein increases were accompanied by ultrastructural changes in cells indicating phenotypic modulation, cells subjected to strain were compared to cells grown under static conditions by transmission electron microscopy (TEM) and fluorescent staining. The strained ASM cells oriented perpendicular to the strain direction were more elongated and contained more actin stress fibers than identical cells grown under physically static conditions. The stress fiber bundles were thicker and reorganized parallel to the long axis of the cell. Marked increases in the numbers and lengths of focal adhesions between the cell membrane and the substratum were found by both TEM and immunostaining for talin. Mechanical strain thus increases organization of cytoskeletal elements in cultured ASM cells. Similar effects in vivo may serve to promote the expression of the contractile phenotype of cultured ASM cells independent of other in vivo factors and alter cell contractility. Increased organization of cytoskeletal elements might also increase the efficiency of signal transduction from the extracellular matrix into the cell interior.