Fibronectin, a glycoprotein present in plasma and extracellular matrix, is believed to be involved in cell-cell and cell-matrix interactions. Using immunofluorescence techniques, we studied the time course of appearance and distribution of fibronectin in healing rabbit corneal epithelial wounds and compared fibronectin to other selected proteins in the cornea. In the normal cornea, fibronectin was detected only in Descemet's membrane and not in the epithelial basement membrane. Shortly after wounding, fibronectin deposited on the denuded corneal surface and was a continuous prominent layer by 8 hours. The epithelium had begun to migrate over the deposited fibronectin by 22 hours and by 52 hours had completely covered the denuded surface. Fibrinogen/fibrin was also detected on the initial bare wound surface. Once the wound was reepithelialized, the subepithelial fibronectin and fibrin layer then progressively disappeared, so that by 2 weeks only a small amount was detected. Fibronectin also appeared in the deep stroma of corneas after wounding in elongated patches, a pattern suggestive of keratocyte association. Fibronectin had an inverse relationship to bullous pemphigoid antigen which was used as a marker for the lamina lucida of the epithelial basement membrane. The bullous pemphigoid antigen, which was found in the normal corneal epithelial basement membrane, was removed with the epithelium and reappeared during the wound healing when fibronectin was diminishing. IgG and albumin did not localize on the wound surface, and the diffuse staining seen in the stroma did not change during healing. These findings are compatible with the hypothesis that fibronectin and fibrin play a role in epithelial migration and temporary adhesion to the surface during corneal wound healing, at a time when the normal anchoring mechanism is lost.