The inner lumen of all blood vessels is comprised of a continuous layer of endothelial cells (ECs). This layer serves as a barrier between flowing blood and other vascular tissues and regulates vessel permeability. The loss of ECs (e.g., due to inflammation or surgery) compromises the integrity of the endothelium and leads to a variety of vascular disorders, especially when the denuded area is not restored in a timely manner. Vascular ECs are constantly exposed to cyclic stretch due to the pulsatile nature of blood flow. Previously we have reported that cyclic stretch inhibits EC motility. Because cell migration is a key element in maintaining an intact EC monolayer and preventing the development of vascular lesions, our findings is consistent with the preferential localization of atherosclerotic lesions at vascular branch sites where wall tension is higher than surrounding vascular segments. We have also shown that there is a two-fold increase in the intracellular reactive oxygen species (ROS) amount in ECs under cyclic stretch. When produced in a controlled manner, ROS function as signal transduction intermediates and are essential for cell migration. However, over production of intracellular ROS has been associated with numerous cardiovascular disorders such as atherosclerosis. In this study, we investigated the relationship between stretch-induced ROS production and stretch-inhibited EC motility. Bovine aortic ECs were seeded on deformable silicone sheets pre-coated with fibronectin. ECs were cultured in Dulbecco's Modified Eagle Medium supplemented with 5% fetal bovine serum at 37oC in a humidified 5% CO2–95% air incubator until they reached confluence. A strip of BAECs was then removed with a cell scraper and the injured EC monolayer was kept as a static control or exposed to cyclic uniaxial stretch (20% elongation strain, 1 Hz) for 3 hours using a custom-designed cell stretching device. Migration of ECs into the denuded area was examined by phase contrast microscopy. Before being subjected to stretch, some cells were pretreated with N-acetyl-L-cysteine (NAC) or diphenylene iodonium (DPI), a ROS scavenger and a flavoprotein inhibitor that blocks a wide spectrum of cellular oxidase systems, respectively. Our results showed that NAC and DPI decreased migration speed in static controls, reaffirming the requisite role of a physiological amount of ROS for EC migration. In stretched samples, NAC and DPI partially improved EC migration speed, suggesting that the inhibitive effect of cyclic stretch on EC motility is at least mediated in part by the excess ROS production.