The phox (phagocyte oxidase) homology (PX) domain occurs in the mammalian phox proteins p40(phox) and p47(phox). the polarity establishment protein Bem1p in budding yeast, and a variety of proteins involved in membrane trafficking. Here we show that the PX domains of p40(phox) and p47(phox) directly bind to phosphoinositides: P40(phox) prefers Ptdlns(3)P, while p47(phox) does Ptdlns(4)P and Ptdlns(3,4)P-2. In addition, the Bem1p PX domain also interacts with Ptdlns(4)P. When the P40(phox) PX domain is expressed as a fusion to green fluorescent protein in HeLa cells, it exists at early endosomes where Ptdlns(3)P is enriched. Furthermore, a mutant P40(phox) PX carrying the substitution of Lys for Arg105 only weakly binds to phosphoinositides in vitro, and fails to locate to early endosomes. Thus the PX domain functions as a novel phosphoinositide-binding module and likely participates in targeting of proteins to membranes.