Involvement of phagocyte NADPH oxidase in host defense response is well established. In contrast, little is known about the functional role of NADPH oxidase in platelets. In this study, we analyzed involvement of platelet NADPH oxidase in aggregation of human platelets and in amplification of production of reactive oxygen species (ROS) by activated human neutrophils. Apocynin, a known NADPH oxidase inhibitor, as well as superoxide dismutase mimetic Mn(III)tetrakis(1-methyl-1-pyridyl)porphyrin, inhibited ROS generation by collagen-activated platelets, collagen-induced aggregation of platelets, as well as collagen-induced release of thromboxane B₂. These data suggest the key role of intracellular ROS derived from NADPH oxidase in the control of thromboxane A₂ (TXA₂) production in platelets stimulated by collagen. Apocynin also inhibited thrombin-induced ROS production and thrombin-induced platelet aggregation. Activation of neutrophils with latex resulted in an outburst of ROS that was inhibited by apocynin. ROS production by latex-stimulated platelets was modest and also inhibited by apocynin. However, when a mixture of platelets and neutrophils was stimulated with latex, ROS production was three to six times higher in comparison with activation of neutrophils alone. Platelet-dependent augmentation of neutrophil ROS production was abrogated by TXA₂ synthase inhibitor (furegrelate, 1 μM) or by aspirin (300 μM). In summary, NADPH oxidase in platelets seems to play a major role as an intracellular signaling mechanism in the activation of platelets. However, in host defense response involving neutrophils and platelets, platelets enhance ROS production by neutrophils and possibly their cytotoxic potential via the release of TXA₂, which in turn in platelets is not affected by the extracellular release of free radicals.