Corneal endothelial wound healing following scrape injury in the rabbit and cat is characterized by cell spreading and maintenance of a normal endothelial phenotype consisting of apically-localized, circumferential microfilament bands and cell border-associated ZO-1, a tight junction protein and marker for endothelial differentiation. In contrast, after freeze injury in the rat and rabbit endothelial cells develop basally organized microfilament bundles (stress fibers), and appear to proliferate and form a multilayered zone at the wound margin. The purpose of the present study was to determine if similar phenotypic changes are observed after freeze injury in the cat corneal endothelium, which like human, normally has limited growth potential. In addition, changes in ZO-1 and alpha-smooth muscle actin (a marker for myofibroblast transformation) distribution were evaluated for the first time following freeze injury. In vivo endothelial healing of standard 3 mm diameter freeze injury was evaluated at 4 hr, 12 hr, 24 hr, 48 hr, 3 days and 5 days after injury in 22 cat eyes. Corneas were stained with phalloidin, propidium iodide, and anti-ZO-1, anti-alpha-smooth muscle-specific actin or anti-fibronectin antibodies. Protein organization was then evaluated using immunofluorescence and laser scanning confocal microscopy. Beginning at 12 hr after injury, endothelial cells appeared to extend and elongate over the wound area. By 48 hr after injury, migrating endothelial cells formed a multilayered activated zone (AZ) at the wound margin. Endothelial cells immediately adjacent to the AZ maintained a normal circumferential organization of f-actin colocalized with cell border-associated anti-ZO-1 staining at all time points observed. However, within the AZ there was an abrupt increase in phalloidin staining and development of prominent microfilament bundles (stress fibers), as well as a loss of normal anti-ZO-1 staining. The AZ also stained positively for anti-alpha-smooth muscle actin and anti-fibronectin antibodies. Changes in the distribution of ZO-1 were observed as early as 4 hr after injury, and appeared to precede f-actin reorganization. These data indicate that endothelial healing after freeze injury in the cat involves a loss of normal endothelial differentiation and cell connectivity, and transformation to a myofibroblastic phenotype.