Paired human donor corneas (age, 73 +/- 12 yr), preserved in organ culture medium, were used to evaluate the effect of human epidermal growth factor (hEGF) on endothelial wound closure rate (WCR), on morphometric parameters (cell size, shape, and density), and on cell division in the wound area. The endothelium of the corneas was mechanically wounded (area, 4.9 +/- 0.9 mm2). For each pair, one cornea was treated with 10 ng/ml hEGF, while the mate served as control. WCR was assessed by daily staining of the corneas with trypan blue. Morphometric data were obtained after alizarin staining. Mitotic activity was assessed using 3H-thymidine autoradiography. Addition of hEGF significantly increased the WCR compared to the control group. In the closed wound (between 4-9 d), the mean cell size in the center averaged 1940 microns2 in the control group and 1287 microns2 in the hEGF-treated group (P less than 0.01). Fifteen days after wounding, the mean cell sizes averaged 1910 microns2 and 1427 microns2 in the control and hEGF-treated group, respectively (P less than 0.01). All corneas exposed to hEGF had higher endothelial cell densities than the control corneas. In the early stages of wound closure, the cells in the transitional zone in hEGF-treated corneas had a somewhat more elongated shape. However, hEGF did not affect the final cell shape within the closed wound. Autoradiographic results revealed that hEGF accelerated DNA-synthesis, although only to a limited extent. The results indicate that, in human corneas, hEGF promotes endothelial wound healing predominantly by cell migration, at least in corneas from senior donors.