Superoxide production by the phagocyte NADPH oxidase is essential for innate immunity as shown in chronic granulomatous disease (CGD), an immunodeficiency disease due to mutations in one of its genes. The NADPH oxidase is composed of two membrane proteins (gp91phox/NOX2 and p22phox) and four cytosolic proteins (p47phox, p67phox, p40phox, and Rac1/2). The phosphorylation of p47phox is required for NADPH oxidase activation in cells. As p47phox and p67phox can form a tight complex in cells, we hypothesized that p67phox could regulate p47phox phosphorylation. To investigate this hypothesis, we used phospho-specific antibodies against five major p47phox-phosphorylated sites (Ser304, Ser315, Ser320, Ser328 and Ser345) and neutrophils from healthy donors and from p67phox-/–CGD patients. Results showed that fMLF and PMA induced the time- and concentration-dependent phosphorylation of p47phox on Ser304, Ser315, Ser320 and Ser328 in healthy human neutrophils. Interestingly, in neutrophils and Epstein-Barr virus-transformed B-lymphocytes from p67phox-/–CGD patients, phosphorylation of p47phox on serine residues was dramatically reduced. In COSphox cells, the presence of p67phox led to increased phosphorylation of p47phox. In vitro studies showed that recombinant p47phox was phosphorylated on Ser304, Ser315, Ser320 and Ser328 by different PKC isoforms and the addition of recombinant p67phox alone or in combination with p40phox potentiated this process. Thus, p67phox and p40phox are required for optimal p47phox phosphorylation on Ser304, Ser315, Ser320 and Ser328 in intact cells. Therefore, p67phox and p40phox are novel regulators of p47phox-phosphorylation.