Endothelial cells (EC) are subjected to hemodynamic forces in vivo. However, most in vitro studies of EC biology have been performed utilizing stationary culture conditions. To study the morphology and cytoskeletal features of EC under dynamic culture conditions, we utilize a system capable of exerting repetitive strain on cells in culture. Human saphenous vein EC were plated to a subconfluent density in 25-mm wells with a thin flexible bottom and a rat collagen, Type I surface. A -20 kPascals vacuum applied to the bottoms led to a maximum deformation of 24%. EC were exposed to 0.5 sec deformation alternating with 0.5 sec relaxation (60 cycles/min) for 24 hr. EC were fixed with formalin at different time intervals and stained with crystal violet. Actin filaments were stained with rhodamine phalloidin, an F-actin marker, while beta-tubulin and vimentin were visualized by immunofluorescent antibody techniques. Within 15 min after initiation of cyclic strain, actin stress fibers were aligned perpendicular to the force vector. By 12 hr of cyclic strain EC were elongated and oriented in the same direction as the actin filaments. EC elongation and alignment were inhibited by cytochalasin B. Even up to 24 hr of cyclic strain, beta-tubulin and vimentin distributions were unaltered. We propose that cyclic strain of EC in vitro influences cell alignment and elongation by a mechanism dependent on the actin filament system.