Activation of the neutrophil respiratory burst oxidase involves phosphorylation-dependent translocation of the cytosolic proteins p47phox and p67phox to the plasma membrane, a process in intact cells that is inhibited by staurosporine. We now report that in a cell-free oxidase system, staurosporine and protein kinase C pseudosubstrate PKC(19-36) both inhibited p47phox phosphorylation but had no effect on superoxide generation. In contrast, p47phox phosphorylation, translocation, and superoxide generation were inhibited by a peptide, p47phox(323-332) (AYRRNSVRFL), based on a putative serine phosphorylation domain. This effect was specific for the 323-332 peptide, as it was not observed with two peptides based on other p47phox phosphorylation domains. All three peptides served as substrates for phosphorylation, but the extent of peptide phosphorylation did not correlate with inhibition of oxidase function. p47phox(325-330), which represents the serine phosphorylation motif of p47phox(323-332), did not inhibit translocation or superoxide production despite its ability to block phosphorylation of p47phox. These data indicate the presence of functionally important sites within the p47phox(323-332) peptide. Although serine 328 is in a consensus phosphorylation motif, the lack of correlation in the cell-free system between p47phox phosphorylation and either protein translocation or superoxide formation suggests that a phosphorylation-independent function resides in the 323-332 segment of p47phox.