The superoxide-generating NADPH oxidase of neutrophils can be activated in a cell-free system consisting of cell membranes, cytosol and an activating detergent (e.g. arachidonate or SDS). It has previously been reported [Aviram and Sharabani (1989) Biochem. Biophys. Res. Commun. 161, 712-719] that a mixture of phosphoinositides (PPIs), as well as the individual inositol lipids, interfere with the activation process. In the present study it is shown that exposure of the cytosol to PPI results in a progressive (t1/2 = 30 s) loss of its oxidase-supporting activity and that Mg2+ ions eliminate this inactivation. Neomycin, previously described as an inhibitor of cell-free activation, counteracted the effect of PPI and vice versa. Fractionation experiments implicated the p67-phox cytosolic component of the oxidase in the association with PPI. PPI blocked activity of recombinant p67-phox also and quenched the fluorescence intensity of its tryptophan residues. It is suggested that PPIs may mediate the interaction of the oxidase with the cytoskeleton and/or with the membrane.