Chymase is a chymotrypsin-like serine protease predominantly produced by mast cells. In this study, human cutaneous and gingival keratinocytes, ovary surface epithelia, and a porcine epithelial cell line were assayed by homology-based cloning and the amplified DNA fragment identified as a chymase. In vitro, chymase could not be induced by serum or cytokine treament alone. Chymase was activated 3X within 60 min in basal media by scratch wounding cultured monolayers and further potentiated over 10X at 18 h by additional serum and cytokine treatment. Chymase activity was cell-associated and found to peak within 24 h of wounding then steadily decreased as cultures healed, reaching baseline levels before confluence was reestablished. Affinity column purified enzyme was found to effectively degrade fibronectin and by Western blot analysis using a human chymase antibody to be of about 30kD. Immunostaining revealed chymase activation at the wound edge colocalizing with reactive oxygen species generation. Specifically, chymase activation was attenuated by inhibition of nitric oxide, superoxide and peroxynitrite. Exogenous peroxynitrite but not hydrogen peroxide also resulted in chymase activation in unwounded monolayers. Disruption of cytoskeletal stress fibers by cytochalasin D attentuated both wound-activated chymase and reactive oxygen species generation. Chymase inhibitor chymostatin reduced the loss of cell-cell contacts and onset of porcine and human skin epithelial cell migration at the wound edge. This shows that an epithelial chymase is rapidly activated by a ligand-independent mechanism following mechanical stress via cytoskeletal and reactive oxygen species signaling and is associated with the onset of epithelial cell migration.