The superoxide-generating NADPH oxidase, dormant in resting phagocytes, is activated during phagocytosis following assembly of the membrane-integrated protein cytochrome b(558) and cytosolic factors. Among the latter are the three proteins containing Src homology 3(SH3) domains,p67(phox),p47(phox) and p40(phox). While the first two factors are indispensable for the activity, p40(phox) is tightly associated with p67(phox) in resting cells and is suggested to have some modulatory role. Here we describe a systematic analysis of the interaction between p40(phox) and p67(phox) using the yeast two-hybrid system and in vitro binding assays with recombinant proteins. Both methods unequivocally showed that the minimum requirements for stable interaction are the C-terminal region of p40(phox) and the region between the two SH3 domains of p67(phox). This interaction is maintained even in the presence of anionic amphiphiles used for the activation of the NADPH oxidase, raising a possibility that it mediates constitutive association of the two factors in both resting and activated cells. The C-terminal region of p40(phox) responsible for the interaction contains a characteristic stretch of amino acids designated as the PC motif, that also exists in other signal-transducing proteins from yeast to human. Intensive site-directed mutagenesis to the motif in p40(phox) revealed that it plays a critical role in the binding to p67(phox). Thus the PC motif appears to represent a novel module for protein-protein interaction used in a variety of signaling pathways.