Fibronectin, which appears at wound sites, serves as a temporary matrix for the epithelial migration involved in healing. Cellular responses to fibronectin have not yet been elucidated. We visualized beta 1 integrins, fibronectin, laminin, and collagen type IV by immunofluorescence techniques at various intervals after single, nonpenetrating incision in the corneas of rats to investigate the chronologic changes in the localization of these proteins during corneal epithelial wound healing. In unwounded corneas beta 1 integrins were found on basal cells of the corneal epithelium and fibronectin was found in the corneal subepithelial region, at Descemet's membrane, and in streaks between collagen fibers of the stroma. Both laminin and collagen type IV were found in the subepithelial region and Descemet's membrane. Immediately after the incision, fibronectin was visible on the surface of the V-shaped defect of the stroma; epithelial cell expressed beta 1 integrins began to migrate over the defect and to fill it. When healing was almost completed, beta 1 integrin staining of epithelial cells diminished, except for in basal cells. Staining of fibronectin diminished at the interface between the new epithelium and the stroma. Laminin and collagen type IV were not seen between the migrating epithelial cells and the underlying stroma until epithelial cells completely covered the defect. Appearance and disappearance of beta 1 integrins and fibronectin are thus well coordinated during corneal epithelial wound healing process, suggesting an active involvement of beta 1 integrins and fibronectin.