Because wounding the epidermis increases proteolytic activity and because disorders associated with barrier dysfunction have elevated protease activity, we studied the effect of protease inhibitors on the time course of barrier recovery and on the development of epidermal hyperplasia induced by repeated injury. After injuries to the epidermis produced by tape stripping, acetone treatment, or detergent (SDS) treatment that disrupt the barrier, a single application of 5% tranexamic acid [4-(aminomethyl)cyclohexane carboxylic acid, t-AMCHA], a well known anti-plasmin reagent, accelerated barrier recovery in both hairless mouse and human skin. In contrast, neither aminocaproic acid nor aminobutyric acid, inactive analogs of t-AMCHA, affected the time course of barrier recovery. Several trypsin-like serine protease inhibitors, e.g., leupeptin, TLCK, and PMSF, also accelerated barrier repair. In contrast other types of protease inhibitors, e.g., EDTA, pepstatin, N-ethylmaleimide, chymostatin, and TPCK, did not accelerate barrier recovery. We next evaluated the effects of daily topical application of t-AMCHA on epidermal hyperplasia, induced by repeated tape stripping or acetone treatment for 7 d. The degree of hyperplasia, quantified by the measurement of epidermal thickness, was reduced in both models by repeated applications of t-AMCHA. Finally, proteolytic activity in both human and mouse epidermis increased 1-2 h after epidermal injuries that disrupt the barrier. These results demonstrate that the inhibition of plasmin, a serine protease, accelerates barrier recovery and inhibits the epidermal hyperplasia induced by repeated barrier disruption, perhaps by decreasing the extent of attendant epidermal injury.