A phagocyte-type NADPH oxidase complex is a major source of endothelial reactive oxygen species (ROS) production, but its biochemical function and regulation remain unclear. In neutrophils, the p47^phox subunit is centrally involved in oxidase activation in response to agonists such as phorbol-12-myristate-13-acetate (PMA). We investigated the role of p47^phox in endothelial cell ROS production in response to PMA or tumor necrosis factor-α (TNFα) stimulation. To specifically address the role of p47^phox, we studied coronary microvascular endothelial cells (CMECs) isolated from p47^phox−/− mice and wild-type controls. p47^phox was absent in hearts of knockout mice whereas the essential oxidase subunit, p22^phox, was expressed in both groups. In the absence of agonist stimulation, the lack of p47^phox did not result in a reduction in NADPH-dependent ROS production in p47^phox−/− CMECs compared with wild-type CMECs. Prestimulation with PMA (100 ng/mL) or TNFα (100 U/mL) for 10 minutes significantly increased NADPH-dependent O₂⁻ production in wild-type CMECs, assessed either by lucigenin (5 μmol/L) chemiluminescence or dichlorohydrofluorescein (DCF) fluorescence. This response was completely lost in p47^phox−/− cells. Transfection of the full-length p47^phox cDNA into p47^phox−/− CMECs caused expression of p47^phox protein and restoration of the O₂⁻ response to PMA and TNFα. In wild-type CMECs, transfection of antisense p47^phox cDNA substantially reduced p47^phox expression and caused loss of the O₂⁻ response to PMA and TNFα. These data show that endothelial cell p47^phox is critical in the upregulation of NADPH oxidase activity by PMA and TNFα.