Tumor necrosis factor (TNF) α may contribute to the deterioration of cardiovascular function in heart failure (HF) through various mechanisms, including the generation of reactive oxygen species (ROS). NADPH oxidase is the major source of ROS in the vascular system, but the interplay between TNFα and NADPH oxidase activation is elusive. As platelets possess NADPH oxidase enzyme, they represent an important tool to investigate the interplay between NADPH oxidase and TNFα in patients with HF. Serum gp91phox (NOX2), the catalytic core of NADPH oxidase, and serum TNFα were measured in 120 HF patients and in 60 healthy subjects. Compared with healthy subjects, HF patients had higher blood levels of NOX2 and TNFα with a progressive increase from NYHA I to NYHA IV classes. NOX2 levels in blood were independently associated with TNFα in HF patients. An in vitro study, performed on platelets from a subgroup of HF patients, shows that TNFα, at concentrations commonly found in HF patients' peripheral circulation, activates platelet NOX2. Thus, TNFα increases ROS production and the extracellular levels of NOX2. These phenomena are inhibited by the NOX2-specific blocking peptide gp91ds-tat. The study provides evidence that circulating NOX2, as well as the activation of NOX2 on platelets, is increased in HF likely as a consequence of the underlying inflammatory process.