Neutrophils play a critical role as a first line of defense against invading pathogens. Recently, a new defense strategy of neutrophils was described, in which pathogens are trapped and killed by NETs. However, the exact underlying mechanisms leading to the formation of NETs remain elusive. Here, we explored the role of the Rac small GTPases in the formation of NETs using neutrophils that lack Rac1, Rac2, or both isoforms. Efficient NET formation was observed in WT and Rac1null neutrophils. In contrast, NET formation was markedly impaired in cells lacking Rac2 or both Rac2 and Rac1. The defect in NET formation in Rac2null cells was rescued by exogenous ROS sources, suggesting that Rac2-mediated ROS generation is required for NET formation. In addition, we assessed the role of NO in NET formation in mouse neutrophils. Blocking NO production with the NOS inhibitor L-NAME significantly reduced NET formation. Moreover, we show that Rac2null cells produce significantly less NO than Rac1null cells or their WT counterparts. Our data suggest that Rac2 is essential for NET formation via pathways involving ROS and NO.