The production of reactive oxygen species (ROS) by neutrophils has a vital role in defence against a range of infectious agents, and is driven by the assembly of a multi-protein complex containing a minimal core of five proteins: the two membrane-bound subunits of cytochrome b 558 (gp91 phox and p22 phox) and three soluble factors (GTP–Rac, p47 phox and p67 phox (refs 1, 2). This minimal complex can reconstitute ROS formation in vitro in the presence of non-physiological amphiphiles such as SDS. p40 phox has subsequently been discovered as a binding partner for p67 phox (ref. 3), but its role in ROS formation is unclear. Phosphoinositide-3-OH kinases (PI (3) Ks) have been implicated in the intracellular signalling pathways coordinating ROS formation 4, 5 but through an unknown mechanism. We show that the addition of p40 phox to the minimal core complex allows a lipid product of PI (3) Ks, phosphatidylinositol 3-phosphate (PtdIns (3) P), to stimulate specifically the formation of ROS. This effect was mediated by binding of PtdIns (3) P to the PX domain 6 of p40 phox. These results offer new insights into the roles for PI (3) Ks and p40 phox in ROS formation and define a cellular ligand for the orphan PX domain.