Platelets participate not only in thrombus formation but also in the regulation of vessel tone, the development of atherosclerosis, angiogenesis, and in neointima formation after vessel wall injury. It is not surprising, therefore, that the platelet activation cascade (including receptor-mediated tethering to the endothelium, rolling, firm adhesion, aggregation, and thrombus formation) is tightly regulated. In addition to already well-defined platelet regulatory factors, such as nitric oxide (NO), prostacyclin (PGI2), and adenosine, reactive oxygen species (ROS) participate in the regulation of platelet activation. Although exogenously derived ROS are known to affect the regulation of platelet activation, recent data suggest that the platelets themselves generate ROS. Intracellular ROS signaling in activated platelets could be of significant relevance after transient platelet contact with the vessel wall, during the recruitment of additional platelets, and in thrombus formation. This review discusses the potential cellular and enzymatic sources of ROS in platelets, their molecular mechanisms of action in platelet activation, and summarizes in vitro and in vivo evidence for their physiological and potential therapeutic relevance.