To understand the mechanisms of epidermal growth factor (EGF)-stimulated re-epithelialization, the roles of microtubules and microfilaments in epithelial migration were investigated using the organ culture of rabbit cornea. The action of EGF was also compared with that of fibronectin. The addition of either EGF or fibronectin increased the epithelial sheet migration. Nocodazole, an inhibitor of the microtubule assembly, alone did not affect the epithelial migration. Nocodazole antagonized the EGF-dependent increase of the epithelial migration when nocodazole coexisted during the period of delivery of the signal from EGF. However, the stimulatory action of fibronectin was observed even in the presence of nocodazole. On the other hand, cytochalasin B, inhibitor of the microfilament assembly, inhibited the epithelial migration in a dose-dependent manner regardless of whether EGF or fibronectin was present or not. These results indicated that the formation of microfilaments was essential for the corneal epithelial migration and that the stimulatory action of EGF on the corneal epithelial migration depended on the assembly of microtubules. Therefore, in corneal epithelial wound healing, the action of EGF requires the organization of both microfilaments and microtubules, while that of fibronectin requires the reorganization of the microfilaments.