This study used video time-lapse recording to characterize the dynamic features of corneal epithelial cells and keratocytes during in vitro wound closure. Confluent cultures of these two cell types from rabbits were established in Rose chambers. A wound 4 or 10 mm in diameter was produced in the center of each culture by mechanical removal of cells. Wound closure was recorded by videomicroscopy for 2-3 days and reviewed at a playback speed of 400 times normal. The epithelial cells at the wound margin initiated migration by extending lamellipodia with undulatory motions. Successive tiers of cells moved as a continuous sheet in a unified and coordinated manner while maintaining intercellular linkage. The migration was unidirectional, toward the wound center. The mean migration rate of the leading cells was 104 microns/hr. The trailing cells migrated at successively slower rates, inversely proportional to their distance from the wound margin. Mitosis was rare during migration but did occur simultaneously. The mitotic rate was 3.7 mitoses/100 cells. The relative mitotic frequency was 0.23 mitosis/hr. By contrast, in keratocyte cultures, the cells around the wound margin migrated individually and asynchronously without intercellular connection. Initially the cells moved generally toward the wound space, but later, different cells migrated in different directions. The mean migration rate was 15 microns/hr. Mitosis occurred frequently. The mitotic rate was 25.3 mitoses/100 cells, and the relative mitotic frequency was 1.33 mitoses/hr. The cell cycle duration was 9.9 hr. Thus corneal epithelial cells and keratocytes showed fundamentally different characteristics and mechanisms of wound closure in vitro.