The maintenance of endothelial monolayer integrity is an important function of aortic endothelial cells. Our study was designed to test two hypotheses related to the repair of the wounded endothelial monolayer. First, that the reappearance of the highly ordered cobblestone monolayer after wound closure is associated with specific sequential changes in the cytoskeletal system. Second, that there are different patterns of reendothelialization depending on whether microfilaments or microtubules are disrupted. One and a half millimeter wide wounds were created down the middle of confluent endothelial monolayers so that there were two wound edges facing each other. During the initiation of repair, the centrosomes of the cells on both sides of the wound reorientated to the front of the cell. The dense peripheral band of actin microfilaments disappeared, the cells elongated and migrated as a uniform sheet with wound closure occurring within 60 hours. The rate of closure remained constant until the migrating fronts met. The cytoskeletal changes observed and the rate of closure were similar to those we reported in a single edge wound. At closure, however, there was a transient piling up of cells which disappeared after 24 to 36 hours. Within 36 to 48 hours after closure, the centrosomes became randomly distributed around the nucleus. By 40 to 48 hours, the dense peripheral band started to reappear and the cells returned to a cobblestone appearance 72 to 96 hours after closure. Thus, the remodeling of the confluent monolayer after wound closure occurs in association with a specific series of cytoskeletal changes. When the microfilaments were disrupted with cytochalasin B, cell migration still occurred but it took up to four times longer for closure. These cells were initially flatter than normal and contained only a few microfilament fibers; however, the microtubule system was intact and centrosome reorientation occurred, but at a slower rate. However, when the microtubules were disrupted with colchicine, either at the onset of wounding or during repair, neither centrosome reorientation nor cell migration occurred. Thus wound closure of small-sized wounds require the presence of intact microtubules, whereas the additional presence of microfilaments results in a more rapid and efficient system of reendothelialzation.