The generation of superoxide anion radicals (O2*-) and the other reactive oxygen species (ROS) was estimated by means of cytochrome c reduction and chemiluminescence, as well in resting blood platelets and in platelets stimulated by thrombin in the presence or absence of some inhibitors of pathways involved in platelet activation. We used allopurinol (xanthine oxidase inhibitor), wortmannin (PI 3-kinase inhibitor) and staurosporine (protein kinase C inhibitor). To determine the involvement of the glutathione in ROS generation, we used L-buthionine sulfoximine (BSO) which blocks GSH synthesis. Our results confirmed that thrombin stimulates the production of ROS concomitant with metabolism of arachidonate and production of malonyldialdehyde (MDA) in blood platelets (P < 0.05) and showed that, in the presence of inhibitors, the generation of ROS in platelets (resting and stimulated) was reduced. This indicates that xanthine oxidase, PI 3-kinase or protein kinase C take part in the formation of ROS in blood platelets. Moreover, adhesion of platelets to fibrinogen and secretion of adenine nucleotides from platelets after wortmannin and staurosporine action was also inhibited. BSO not only decreased GSH level, but also reduced the amount of ROS; a correlation between the depletion of GSH and the decrease of ROS was observed (R = -0.987; P < 0.02). It is concluded that in blood platelets, ROS are produced in the receptor-mediated signaling pathways and platelet activation (arachidonic acid metabolism, the glutathione cycle, metabolism of phosphoinositoides and due to xanthine oxidase). Our results support the importance of ROS in platelet function.