The role of plasminogen activator (PA) in the migration of corneal reepithelialization was studied. Rabbit corneal blocks were cultured, and both the extent of epithelial migration over the exposed corneal stroma and the activity of PA released into the culture media were measured. A significant, direct correlation between epithelial migration and PA activity in the medium was observed, even when the migration was stimulated by fibronectin or EGF, or was inhibited by cytochalasin B or cycloheximide. Zymography confirmed that the PA released into the culture medium was of the urokinase type (u-PA). Immunohistochemical studies showed that u-PA and plasmin(ogen) were present at the leading edge of the migrating epithelium. Studies of corneal cell cultures indicated that epithelial cells rather than endothelial cells or fibroblasts were the source of the u-PA. The addition of antihuman u-PA IgG or protease inhibitors retarded the migration of the corneal epithelium in a dose-dependent manner, indicating that u-PA activity is essential for the migration of the corneal epithelium. These findings suggest that the migration of corneal epithelial cells requires not only cell attachment to the extracellular matrix through the fibronectin but also degradation of the fibronectin by the release of cellular u-PA.