Eosinophils contain many cytotoxic mediators including eosinophil peroxidase (EPO) in their granules; on degranulation, these mediators are released by various pathophysiological stimuli, resulting in severe tissue damage. However, little is known about inhibitors of degranulation. Here, we report that eosinophils isolated from patients with bronchial asthma have significant chymotrypsin-like serine protease activity in the high salt extract fraction. The protease partially purified and labeled with [3H]diisopropylfluorophosphate has an apparent molecular mass of 28 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chymotrypsin-like protease was not immunoreactive with antibodies against chymase and atypical chymase from rat mast cells or with an antibody against cathepsin G from human neutrophils. Studies on the subcellular distribution of the chymotrypsin-like protease indicated that the enzyme is mainly localized with EPO in eosinophil granules. Chymostatin, an inhibitor of the chymotrypsin-like protease(s), but not an inhibitor of other types of proteases, markedly inhibited the EPO release from eosinophils that was induced by immunoglobulin G plus rIL-5 or platelet-activating factor, although it had no effect on the release of EPO induced by the calcium ionophore A23187. These results suggest that proteolytic activation by chymotrypsin-like serine protease(s) in eosinophils plays some role in the process of receptor-mediated EPO release from the granules.