Human platelets can be stimulated by recombinant human fifth component of complement (rhC5a) in the presence of human neutrophils. After challenge with N-formyl-Met-Leu-Phe or rhC5a, concentrated neutrophils release cathepsin G into the supernatant. The concentrations of cathepsin G recovered by titration of the enzymatic activity correlate with the capability of these supernatants to induce platelet stimulation as measured by serotonin release. Cathepsin G purified from neutrophil granules triggered platelet aggregation and serotonin release independent of arachidonic acid metabolites and platelet-activating factor formation. A concentration of 100 nM of cathepsin G, which was reached in the surrounding space of activated neutrophils, induced a 50% platelet stimulation. Three distinct antiproteinases were tested against cathepsin G-induced platelet activation. Z-Gly-Leu-Phe-CH2Cl, a specific inhibitor of cathepsin G enzymatic activity, proved to be nonspecific in our biological system. By contrast, alpha 1-antichymotrypsin and alpha 1-antitrypsin displayed specific activities. The physiological specific inhibitor of cathepsin G, alpha 1-antichymotrypsin, was the most potent and was used in the rhC5a-induced neutrophils-mediated platelet activation. A complete inhibition was achieved, showing that release of cathepsin G from neutrophils accounts for platelet activation. Such a chain of events involving C5a, neutrophils, cathepsin G, and platelets may be of relevance in certain inflammatory states, particularly the adult respiratory distress syndrome.