Rho and Rac, two members of the Ras superfamily of guanosine triphosphate (GIP)-binding proteins, regulate a variety of signal transduction pathways in eukaryotic cells. Upon stimulation of phagocytic cells, Rac enhances the activity of the enzyme nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) oxidase, resulting in the production of superoxide radicals. Activation of the NADPH oxidase requires the assembly of a multimolecular complex at the plasma membrane consisting of two integral membrane proteins, gp91(phox) and p2l(phox), and two cytosolic proteins, p67(phox) and p47(phox). Rac1 interacted directly with p67(phox) in GTP-dependent manner. Modified forms of Rac with mutations in the effector site did not stimulate oxidase activity or bind to p67(phox). Thus, p67(phox) appears to be the Rac effector protein in the NADPH oxidase complex.