p67(PHOX), a cytosolic component of the NADPH oxidase complex, is phosphorylated during neutrophil activation by several agonists. The intracellular signaling pathways leading to its phosphorylation in neutrophils may involve a PKC-dependent pathway and a PKC-independent pathway. Here, we analyzed p67(PHOX) phosphorylation by ERK2 and p38MAPK. Both ERK2 and p38MAPK phosphorylated p67(PHOX) in vitro, with similar K(m) values (10 and 9 microM, respectively). Phosphopeptide mapping indicated that ERK2 and p38MAPK phosphorylate different subgroups of peptides. Using truncated forms of p67(PHOX), we found that the major phosphorylation target site of ERK2 was located in the N-terminal fragment (1-243), while the major phosphorylation target sites of p38MAPK were located in the C-terminal fragment (244-526). Furthermore, an additional peptide, which was not phosphorylated in the intact protein, appeared to be phosphorylated in the isolated C-terminal fragment (aa 244-526). This site may not thus be accessible in the intact protein. Indeed, incubation of the C-terminal fragment (244-526) with different N-terminal fragments (1-243, 1-210, or 1-199) containing the tetratricopeptide-rich region prevented phosphorylation of this C-terminal fragment. ERK1/2 and p38MAPK are also involved in p67(PHOX) phosphorylation in intact neutrophils. Indeed, PD98059 and SB203580, two selective inhibitors of MEK1/2 and p38MAPK, respectively, inhibited p67(PHOX) phosphorylation in fMLP- and PMA-stimulated neutrophils, with additive effects, thus suggesting that they also target different sites in vivo. Furthermore, the major peptides phosphorylated by ERK2 and p38MAPK in vitro were also phosphorylated in fMLP-stimulated neutrophils. Taken together, these results suggest not only that p67(PHOX) is phosphorylated by ERK2 and p38MAPK in vitro and in intact neutrophils on several selective sites but also that a C-terminal phosphorylation site may become accessible after a conformational change of the protein.