The endothelial cell is unique because it must undergo a transition from a resting cell with a cytoskeleton organized for barrier function to one which promotes cell translocation following denuding endothelial injury. Since actin microfilaments are critical for both maintaining the integrity of the resting monolayer and for optimum reendothelialization, we carried out a detailed study of the organization of microfilaments as the cell undergoes the transition from a resting to a translocating cell. We used an in vitro model in which a linear wound was made in a confluent monolayer of porcine aortic endothelial cells. The complex reorganization of actin microfilament bundles following injury and their relationship to microtubules and vinculin was studied in cells at the wound edge using immunofluorescent scanning laser confocal microscopy and time-lapse videomicroscopy. In the resting confluent monolayer, microfilaments were present as a dense peripheral band (DPB) located toward the upper part of the cell and as central microfilament bundles at the substratum. Three distinct stages of microfilament reorganization occurred sequentially during early repair. Stage 1 followed wounding and involved the reduction of the DPBs of microfilaments and associated peripheral cell-cell vinculin plaques. This was associated with rapid forward actin-based lamellipodia extrusions and cell elongation. Low-dose cytochalasin, which did not disrupt the morphology of microfilament bundles, reduced elongation. Stage 2 was characterized by central microfilaments behind the lamellipodia distributed parallel to the wound edge with vinculin plaques at their tips. This was associated with prominent spreading at the front of the cell which enhanced the extent of coverage of the denuded wound area. Stage 3 was characterized by the orientation of central microfilaments perpendicular to the wound edge with vinculin plaques at their tips and was associated with the initiation of cell translocation. There was no specific structural association between central microfilaments and microtubules as the former were toward the substratum while the latter were toward the center and upper part of the cell. Thus, the sequential appearance of three patterns of microfilament distribution define the cytoskeletal events that regulate the reestablishment of endothelial integrity following denuding endothelial injury.