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-alpha (TNFalpha) 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 TNFalpha (100 U/mL) for 10 minutes significantly increased NADPH-dependent O(2)(-) production in wild-type CMECs, assessed either by lucigenin (5 micromol/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 O(2)(-) response to PMA and TNFalpha. In wild-type CMECs, transfection of antisense p47(phox) cDNA substantially reduced p47(phox) expression and caused loss of the O(2)(-) response to PMA and TNFalpha. These data show that endothelial cell p47(phox) is critical in the upregulation of NADPH oxidase activity by PMA and TNFalpha.