In order to verify if H₂O₂ affects platelet function, platelet-rich plasma and human washed platelets were incubated with subthreshold concentrations (STC) of collagen or arachidonic acid or ADP and/or with 75–150 μ<i>M</i> H₂O₂. While H₂O₂ alone did not affect platelet aggregation, it amplified platelet aggregation response in samples stimulated with STC of arachidonic acid and collagen but not in samples stimulated with STC of ADP. When platelets were preventively treated with aspirin, a cyclooxygenase inhibitor, the platelet activation by H₂O₂ was not observed. Thromboxane A₂ (TxA₂) was not produced by human washed platelets stimulated with STC of arachidonic acid, collagen or by H₂O₂ alone. On the contrary, when STC of agonists were tested on platelets supplemented with H₂O₂ an evident TxA₂ production was seen. This effect was prevented by aspirin pretreatment or by the addition of catalase, an enzyme which destroys H₂O₂. This study suggests that H₂O₂ triggers the activation of platelets exposed to STC of collagen and arachidonic acid, via the cyclooxygenase pathway.