Collagenase is secreted from neutrophils as a latent or proenzyme. In an effort to understand the mechanism of collagenase activation in inflammation, human peripheral neutrophils (PMNs) were isolated and incubated with the tumor promotor, phorbol myristate acetate (PMA), which induces the neutrophils to degranulate and secrete proteinases. Neutrophil media were then treated with various activators or inhibitors of collagenase and other proteinases, and the collagenase activity was measured. A serine proteinase secreted from neutrophils, cathepsin G, was found to activate latent collagenase, but it was also found to require activation itself. Both hypochlorous acid (HOCl) and oxidized glutathione (GSSG) were tested for their collagenase-activating ability and were found to be successful only in the presence of active cathepsin G. A specific cathepsin G inhibitor (0.5 mM Z-Gly-Leu-Phe-CH2Cl) prevented the activation of latent collagenase by HOCl. To confirm these results, purified neutrophil cathepsin G was incubated with a neutrophil proteinase mixture which contained latent collagenase. The collagenase was shown to be activated upon incubation with purified cathepsin G. These results indicate that cathepsin G is a key mediator in neutrophil collagenase activation.