Phosphorylation of p47 phox is a key event in NADPH oxidase activation. We examined the ability of proinflammatory cytokines such as TNFα, IL-1, and G-CSF to induce this process compared with GM-CSF. Only TNF-α and GM-CSF induced a clear p47 phox phosphorylation. This phosphorylation was time dependent and reached its maximum at 20 min. Two-dimensional phosphopeptide mapping of p47 phox phosphorylated in neutrophils primed with TNF-α revealed partial phosphorylation of p47 phox on the same peptide as for GM-CSF. Neutrophil incubation with TNF-α and subsequent addition of the chemotactic peptide fMLP resulted in more intense phosphorylation of p47 phox sites than with each reagent alone. A neutralizing Ab against the p55 TNF receptor, contrary to a neutralizing Ab against the p75 TNF receptor, inhibited TNF-α-induced p47 phox phosphorylation. Neutrophil treatment with both TNF-α and GM-CSF resulted in more intense phosphorylation of the same p47 phox peptide observed with each cytokine alone, suggesting that they engaged pathways converging on common serines. This additive effect was also obtained on the priming of NADPH oxidase activity. The use of protein kinase inhibitors pointed to the involvement of a protein tyrosine kinase, but not protein kinase C. These findings show that TNF-α, via its p55 receptor, induces a protein tyrosine kinase-dependent selective phosphorylation of p47 phox on specific serines. The ability of TNF-α and GM-CSF, two different cytokines with two different receptors to induce this specific p47 phox phosphorylation, suggests that this event could be a common element of the priming of neutrophils by TNF-α and GM-CSF.