We have previously demonstrated that cathepsin G is a strong platelet agonist. However, the ability of cathepsin G to function in this capacity in vivo has remained speculative because the enzyme might be expected to be rapidly neutralized by the high concentration of circulating plasma antiproteases. To examine the physiological significance of cathepsin G as a paracrine mediator, indo-1 and ¹⁴C-5-hydroxytryptamine-loaded platelets were incubated with autologous unloaded neutrophils specifically activated by addition of fMet-Leu-Phe. FMet-Leu-Phe induced substantial increases in cytosolic calcium and 5-hydroxytryptamine release even in the presence of increasing amounts of citrated plasma, indicating that cathepsin G can stimulate platelets under conditions similar to those that may be encountered in vivo. Platelet stimulation was abolished by addition of α1-antichymotrypsin, demonstrating that cathepsin G was the neutrophil mediator responsible for cell activation. Having obtained evidence that cathepsin G can function in the presence of plasma, we measured its ability to hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdIns4,5-P2) and generate phosphatidic acid (PtdA) in aspirin-treated platelets. Our previous observations suggested that cathepsin G stimulates phospholipase C since the protease induces an elevation in [Ca²⁺]i in the presence of exogenous EGTA. Within 10 s of stimulation cathepsin G induced a transient loss in [³²P]-PtdIns4,5-P2 and a concurrent increase in [³²P]-PtdA. [³²P]-PtdA formation was increased over 15-fold in a concentration-dependent manner by cathepsin G. We also determined that cathepsin G induces the release of the lysosomal enzyme β-N-acetyl-glucosaminidase. Both the increase in PtdA and the release of β-hexoseaminidase were comparable to responses elicited by thrombin. These results provide additional evidence that cathepsin G is a strong platelet agonist, support the conclusion that cathepsin G stimulates phospholipase C, and clearly suggest that cathepsin G can function as an agonist in vivo.