We investigated the effect of bacterial toxins that modify and inactivate Rho GTP-binding proteins on the migratory response of endothelial cells to wounding. C3-transferase from Clostridium botulinum, EDIN from Staphylococcus aureus, and toxin A from Clostridium difficile blocked migration of human umbilical vein endothelial cells (HUVECs) in an in vitro wound repair assay. Migrating HUVECs expressed actin microspikes (maximum at 10 minutes after wounding), ruffles (maximum at 12 hours), and fibers (maximum at 24 hours), and within these actin structures, vinculin-containing focal complexes/adhesions were formed. C3-Transferase ADP ribosylated RhoA, RhoB, and RhoC in HUVECs and abolished the formation of actin stress fibers/focal adhesions but had no effect on expression of microspikes, ruffles, or the associated vinculin-containing focal complexes. Similar results were obtained with EDIN and toxin A. These results indicate that endothelial cells migrating into a wounded area express distinct combinations of actin/vinculin structures in a spatially and temporally coordinated manner. The GTPase Rho selectively controls the formation of actin fibers/focal adhesions that occurs 2 to 24 hours after wounding. A mechanism is proposed by which Rho-specific bacterial toxins could influence vascular repair, angiogenesis, or atherosclerosis.