We investigated the effects of two calmodulin inhibitors, trifluoperazine and N-6-aminohexyl-5-chloro-l-naphthalenesulfonamide, on corneal epithelial wound closure in rabbits and rats. Measured, round epithelial defects were made on corneas by gentle scraping. After 20 h in organ culture with or without inhibitor, the remaining wound area was measured and samples were fixed for microscopy. In the rabbit, these inhibitors had little or no effect on the rate of wound coverage. In the rat, however, both trifluoperazine (3–40 μ M) and N-6-aminohexyl-5-chloro-l-naphthalenesulfonamide (10–100 μ M) inhibited wound closure in a dose-dependent manner. Thus we believe that calmodulin plays a crucial role in epithelial migration in the rat. In the rabbit, there seems to exist a mechanism that is not critically dependent upon an intact calmodulin pathway.