Plant cells produce reactive oxygen species (ROS) in response to many stimuli. However, the mechanism of ROS biosynthesis remains unclear. We have explored the hypothesis that the superoxide burst in plants mechanistically resembles the oxidative burst in neutrophils. First we have confirmed that ROS production, which occurs in suspension-cultured soybean (Glycine max) cells in response to hypo-osmotic shock, is inhibited by diphenylene iodonium, an inhibitor of the flavin-dependent oxidase of neutrophils. Because a Rac family G protein is an essential regulator of this NADPH oxidase, and because many plant homologs of Rac have been cloned, we next examined whether Rac-like proteins might be involved in the oxidative burst in the soybean cells. We identified a Rac-like 21-kD soybean protein that cross-reacts with antibodies to human Rac and garden pea Rop and also binds [γ-35S] GTP, a diagnostic trait of small G proteins. This Rac-related protein translocated from the cytosol to microsomes during the oxidative burst. Moreover, soybean cells transiently transformed with either a dominant negative (RacN17) or a dominant positive (RacV12) form of Rac1 showed the anticipated altered responses to three different stimuli: hypo-osmotic shock, oligo-GalUA, and harpin. In response to these stimuli, cells transformed with RacN17 produced less ROS and cells transformed with RacV12 generated more ROS than control cells. These results strongly suggest that a Rac-related protein participates in the regulation of ROS production in soybean cells, possibly via activation of an enzyme complex similar to the NADPH oxidase of phagocytes in animal systems.