OBJECTIVE To examine whether the topical application of keratinocyte growth factor (KGF) can enhance corneal epithelial healing in vivo. In addition, the distribution of S-phase cells in KGF-treated and control corneas was investigated during regeneration and under normal conditions. METHODS A 10-mm diameter epithelial defect was made in the center of rabbit corneas. A 50-microliters aliquot of 10 micrograms/ml human keratinocyte growth factor (hKGF) was then applied topically five times a day. The same volume of phosphate-buffered saline (PBS) vehicle was applied to the contralateral eye as a control. Each corneal epithelial defect was subsequently photographed every 12 hours and was measured by a computer-assisted digitizer. For the S-phase cell analysis, entire corneas were labeled with 3H-thymidine and were subjected to autoradiography at 24 hours after wounding or in the normal cornea at 24 hours after the application of KGF or PBS. RESULTS Topical application of 10 micrograms/ml hKGF significantly accelerated corneal epithelial wound healing when compared with controls. In the S-phase cell analysis, KGF did not have any effect on normal corneal epithelial cells. However, in the regenerating cornea, the number of S-phase cells in the KGF-treated limbal epithelium was twofold higher than in the controls. CONCLUSIONS Topical application of KGF accelerated corneal epithelial wound healing in vivo and increased cell proliferation in the limbal epithelium of the regenerating cornea.