We have found previously that fibronectin enhances the migration of rabbit corneal epithelium both in vitro and in vivo. In this paper we report a change of actin localization in migrating corneal epithelium as determined by immunofluorescent microscopy. Rabbit cornea was cut into small blocks and cultured in TC-199 medium. In the normal cornea, actin was detected as diffuse fluorescence at each epithelial layer. After 8 hr of cultivation epithelial cells had not started to migrate significantly, but actin had accumulated at the cell membrane. After 24 hr, epithelial migration had begun, and actin-specific fluorescence was detected mainly in the basal cell layer at the leading edge. When fibronectin or epidermal growth factor was added to the culture medium, epithelial migration began 8 hr after initiation of culture, and at 24 hr actin-specific fluorescence at the basal side of the migrating epithelial cells appeared stronger than that of a control group cultured in TC-199 unsupplemented medium. At the same time, fibronectin-specific fluorescence was more intense beneath the migrating epithelial cells. It is known that fibronectin has an affinity to collagen, and thus it might coat the cut surface of the stroma. Epithelial cells may attach then to the stroma via coated fibronectin. When a large quantity of exogenous fibronectin is added or when fibronectin is synthesized by the addition of epidermal growth factor, it may further stimulate the organization of intracellular actin from globular form (G-actin) to fibrilar form (F-actin). As a result, the change of intracellular localization and appearance of organized actin molecule might lead to cellular migration.