Transforming growth factor-beta (TGF-beta) is a unique polypeptide growth factor which has been found to stimulate mesenchymal cell proliferation and inhibit epithelial cell growth. Whether or not TGF-beta inhibits corneal epithelial wound closure remains controversial. This study evaluated the effects of TGF-beta on corneal epithelial wound healing in vitro. Rabbit corneas with an artificial 8 mm epithelial defect were organ-cultured for 48 hr in TC-199 supplemented with TGF-beta (100 pg/ml, 1 ng/ml, or 10 ng/ml). The contralateral eye served as control. TGF-beta at 1 ng/ml and 10 ng/ml concentrations showed significant decrease in wound-healing rate compared with the control. 5-bromo-2'-deoxyuridine-immunohistochemistry showed a significant decrease in S-phase cell count in the 1 ng/ml group compared with the control. These results strongly suggest that TGF-beta inhibits corneal epithelial wound healing.