NADPH oxidase is a plasma membrane enzyme of phagocytes generating superoxide anions which serve as bactericidal agents. Activation of this multimolecular enzyme minimally requires assembly at the membrane with flavocytochrome b558 of cytosolic components p47phox, p67phox, and Rac proteins. Rac1 and Rac2 are 92% homologous cytosolic small GTPase proteins. Both Rac1 and Rac2 have been implicated with NADPH oxidase activation in vitro; however, Rac2 is largely predominant in human phagocytes. Here, using the yeast two-hybrid system, we provide data demonstrating in vivo interactions between human p47phox, p67phox, and Rac proteins. Rac proteins interact with p67phox in a GTP-dependent manner, but do not interact with p47phox. Moreover, Rac effector site mutants, which are known to be inactive in NADPH oxidase, lose their interaction with p67phox; Rac2L61 mutant, which has an increased NADPH oxidase affinity, shows an increased affinity for p67phox. Finally, we observe that p67phox interacts 6-fold better with Rac2 than with Rac1. We also show a strong intracellular interaction between p47phox and p67phox. These results indicate that activated Rac can regulate NADPH oxidase by interacting with p67phox and that Rac2 is the main p67phox-interacting GTPase in human cells.