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We studied the association between the production of reactive oxygen species, actin organization, and cellular motility. We have used an endothelial cell monolayer-wounding assay to demonstrate that the cells at the margin of the wound thus created produced significantly more free radicals than did cells in distant rows. The rate of incorporation of actin monomers into filaments was fastest at the wound margin, where heightened production of free radicals was detected. We have tested the effect of decreasing reactive oxygen species production on the migration of endothelial cells and on actin polymerization. The NADPH inhibitor diphenylene iodonium and the superoxide dismutase mimetic manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) virtually abolished cytochalasin D-inhibitable actin monomer incorporation at the fast-growing barbed ends of filaments. Moreover, endothelial cell migration within the wound was significantly retarded in the presence of both diphenylene iodonium and MnTMPyP. We conclude that migration of endothelial cells in response to loss of confluence includes the intracellular production of reactive oxygen species, which contribute to the actin cytoskeleton reorganization required for the migratory behavior of endothelial cells.