Although oxidant generation by NADPH oxidase is known to play an important role in signaling in endothelial cells, the basis of activation of NADPH oxidase is incompletely understood. The atypical isoform of protein kinase C, PKCζ, has been implicated in the mechanism of tumor necrosis factor-α (TNF-α)–induced oxidant generation in endothelial cells; thus, in the present study, we have addressed the role of PKCζ in regulating NADPH oxidase function. We showed by immunoblotting and confocal microscopy the presence of the major cytosolic NADPH oxidase subunits, p47^phox and membrane-bound gp91^phox in human pulmonary artery endothelial (HPAE) cells. TNF-α failed to activate oxidant generation in lung vascular endothelial cells derived from p47^phox−/− and gp91^phox−/− mice, indicating the requirement of NADPH oxidase in mediating the oxidant generation in endothelial cells. Stimulation of HPAE cells with TNF-α resulted in the phosphorylation of p47^phox and its association with gp91^phox. Inhibition of PKCζ by multiple pharmacological and genetic approaches prevented the TNF-α–induced phosphorylation of p47^phox, and its translocation to the membrane. PKCζ was shown to colocalize with p47^phox, and inhibition of PKCζ activation prevented the interaction of p47^phox with gp91^phox induced by TNF-α. Furthermore, inhibition of association of p47^phox with gp91^phox prevented the oxidant generation in endothelial cells. These data demonstrate a novel function of PKCζ in signaling oxidant generation in endothelial cells by the activation of NADPH oxidase, which may be important in mediating endothelial activation responses.