The Rac guanosine 5’-triphosphate (GTP)-binding proteins regulate oxidant production by phagocytic leukocytes. Two Ste20-related p21-activated kinases (PAKs) were identified as targets of Rac in human neutrophils. Activity of the similar to 65- and similar to 68-kilodalton PAKs was rapidly stimulated by chemoattractants acting through pertussis toxin-sensitive heterotrimeric GTP-binding proteins (G proteins). Native and recombinant PAKs phos phorylated the p47(phox) reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase component in a Rac-GTP-dependent manner. The action of PAKs during phagocyte activation by G protein-coupled pathways may contribute to regulation of NADPH oxidase activity.