The influence of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF) I and II on wound healing was investigated in a corneal endothelial system with minimal mitotic activity, using serum-deprived kitten corneal endothelial-cell cultures. After wounding, growth factors were added and wound diameter was evaluated. The DNA synthesis was determined by 3H-thymidine labeling. Wounds did not close in the control cultures grown in serum-free medium without growth factors. The IGF I or II, alone (10 and 100 ng/ml) or added (10 ng/ml) to EGF or bFGF, had no significant effect on wound closure or thymidine uptake. With EGF or bFGF (10 ng/ml), wounds closed after 15 days. Wounds closed after 10 days with EGF or bFGF (100 ng/ml) alone or with the combination of EGF and bFGF (each at 10 ng/ml). Combined EGF and bFGF (each at 100 ng/ml) did not enhance wound closure further. Thymidine uptake was significantly higher in cultures treated with EGF or bFGF (10 ng/ml) than in controls. The uptake could be increased, if both growth factors were combined, but only to the same level achieved with a single factor at 100 ng/ml. This study showed that EGF and bFGF, but not IGF I or II, enhanced wound closure and DNA synthesis in a corneal endothelial cell system that had minimal mitotic activity.