Human corneal endothelial cells (HCEC) do not mitose extensively in vivo after damage to the endothelial layer. However, HCEC will divide in vitro if cultured under appropriate conditions. We measured the ability of various sera, plasma, growth factors, and nutritional substances to stimulate mitosis of HCEC during 5 days of organ culture after a central freeze injury to the endothelium. Supplementation of a chemically defined medium (CDM) with 20% fetal human serum (FHS) induced significantly higher numbers of mitotic figures or labeled nuclei of human or cat corneas compared with paired corneas cultured in CDM alone. Furthermore, addition of 20% FHS produced more labeled nuclei than did addition of 20% fetal bovine serum or 20% adult human serum. Dialyzed fetal human serum failed to stimulate mitosis, indicating that one or more components of fetal human serum with molecular weight <12,000 are essential for mitosis. Human plasma also failed to stimulate mitosis, but an extract of human platelets significantly stimulated high levels of nuclear labeling, suggesting that growth factors contained in platelet granules were responsible for serum-stimulated mitosis of HCEC. Addition of 100 nM epidermal growth factor (EGF) or 10 µM insulin to CDM supplemented with low levels of adult human serum (0.5%) stimulated significantly higher numbers of labeled nuclei compared with paired corneas cultured with 0.5% adult human serum. Supplementation of corneal storage media (K-Sol and CSM) with a mixture of chemically defined agents consisting of EGF, insulin, transferrin, selenium, linoleic acid, and albumin stimulated significantly higher numbers of labeled nuclei compared with paired corneas cultured in the unsupplemented corneal storage media. These results demonstrate that HCEC can be stimulated to undergo extensive mitosis in vitro in response to a combination of chemically defined agents, and suggest that it may be possible to enhance healing of human endothelial cell injuries in vivo or during corneal storage by exposure to appropriate mitogenic factors.