Endothelial damage is an early step in atherosclerotic lesion formation, which is enhanced by pro-atherogenic inhibition of endothelial repair. Previous in vitro studies have suggested a role for increased production of oxygen free radicals in endothelial damage repair. Since cigarette smoke extracts induce oxidative stress, we have examined the potential role of additional smoke-induced oxidative stress in mediating inhibition of endothelial repair in a migration (scratch wound) assay. Methods. Confluent monolayers of human umbilical vein endothelial cells (HUVEC) were scratched with a pipette tip, creating a ~800µm wound. Migration of HUVEC across the wound was assessed over 20 hours by image capture and computer analysis of wound width using the IncuCyte platform (Essen Instruments). Cigarette smoke total particulate matter (TPM) from University of Kentucky 3R4F reference cigarettes was trapped on a Cambridge filter pad, eluted in DMSO, and added to cells immediately after wounding. Free radical production was examined by loading cells with the non-fluorescent indicator dyes 2’,7’ dichlorofluorescein (DCFH) or dihydroethidine (DHE) 4 hours after wounding and measuring free radical generated fluorescent dye products by fluorescence microscopy. In experiments with ascorbic acid, cells were incubated with the antioxidant both for 5 hours prior to and during exposure to TPM. Results. Contrary to previous reports, fluorescence emitted from cells loaded with DCFH or DHE was not greater at the wound edge when compared to cells located away from the wound. (Table 1). Incubation of cells with the antioxidant L-ascorbic acid alone (200µM) had no effect on endothelial migration in the endothelial wound repair assay and was also without effect on the ability of TPM to inhibit endothelial repair in this assay (Table 2). Conclusion. Our data suggest that oxidative stress is unimportant in endothelial wound repair in vitro, and may argue against a role for oxidative stress in the inhibition of endothelial wound repair caused by cigarette smoke extracts.