In this study, we investigated whether (1) collagen-induced platelet aggregation is associated with a burst of H₂O₂, (2) this oxidant species is involved in the activation of platelets, and (3) the pathways of platelet activation are stimulated by H₂O₂. Collagen-induced platelet aggregation was associated with production of H₂O₂, which was abolished by catalase, an enzyme that destroys H₂O₂. H₂O₂ production was not observed when ADP or thrombin were used as agonists. Catalase inhibited dose-dependently thromboxane A₂ production, release of arachidonic acid from platelet membrane, and Inositol 1,4,5P₃ (IP3) formation. In aspirin-treated platelets stimulated with high concentrations of collagen, catalase inhibited platelet aggregation, calcium mobilization, and IP3 production. This study suggests that collagen-induced platelet aggregation is associated with a burst of H₂O₂ that acts as a second messenger by stimulating the arachidonic acid metabolism and phospholipase C pathway.