Purpose.To evaluate for the first time cell junctional protein organization in proliferating corneal endothelial cells during in vivo wound healing.Methods.A total of 16 cats (32 eyes) were used in this study. A single 3-mm diameter (n= 24) or 1-to 2-mm diameter (n= 8) scrape injury was created in the central corneal endothelium of each eye. Twenty-four, 48, 72 hours or 5 days after scrape injury, eyes were collected for in situ double-or triple-labeling with phalloidin, anti-ZO-1, &agr;-catenin, &bgr;-catenin, and MIB-1 (monoclonal antibody to Ki67, a marker for actively cycling cells) and were imaged using confocal laser microscopy.Results.In 3-mm diameter injuries, endothelial cells completely resurfaced the wound 48 to 72 hours after scrape injury; smaller wounds resurfaced by 48 hours. Ki67 staining was negative 24 hours after scrape injury in all cases. Ki67-positive cells were observed in the central region of the wounds after 48 and 72 hours, and mitotic figures and pairs of postmitotic cells were observed. On day 5, Ki67-positive cells were rarely detected, and no mitotic figures were observed. In the wound area, a significant increase in cell area and a reduction in hexagonality were observed in cycling cells after 48 and 72 hours. Normal apical, pericellular staining of f-actin, ZO-1, &agr;-catenin, and &bgr;-catenin was partially maintained at all times during wound healing of small and large wounds. Double-labeling confirmed that these proteins were also present along the apical cell border in Ki67-positive cells.Conclusions.After in vivo scrape injury, proliferation is limited temporally and spatially to spreading endothelial cells within the wound. Cell junctional connections appear to be maintained in actively cycling cells during healing.