Rac1 has been implicated in the generation of reactive oxygen species (ROS) in several cell types, but the enzymatic origin of the ROS has not been proven. The present studies demonstrate that Nox1, a homolog of the phagocyte NADPH-oxidase component gp91 phox , is activated by Rac1. When Nox1 is co-expressed along with its regulatory subunits NOXO1 and NOXA1, significant ROS generation is seen. Herein, co-expression of constitutively active Rac1(G12V), but not wild-type Rac1, resulted in marked further stimulation of activity. Decreased Rac1 expression using small interfering RNA reduced Nox1-dependent ROS. CDC42(G12V) failed to increase activity, and small interfering RNA directed against CDC42 failed to decrease activity, pointing to specificity for Rac. TPR domain mutants of NOXA1 that interfere with Rac1 binding were ineffective in supporting Nox1-dependent ROS generation. Immunoprecipitation experiments demonstrated a complex containing Rac1(G12V), NOXO1, NOXA1, and Nox1. CDC42(G12V) could not substitute for Rac1(G12V) in such a complex. Nox1 formed a complex with Rac1(G12V) that was independent of NOXA1 and NOXO1, consistent with direct binding of Rac1(G12V) to Nox1. Rac1(G12V) interaction with NOXA1 was enhanced by Nox1 and NOXO1, suggesting cooperative binding. A model is presented comparing activation by regulatory subunits of Nox1 versus gp91 phox (Nox2) in which Rac1 activation provides a major trigger that acutely activates Nox1-dependent ROS generation.Rho family GTPases are implicated in innate immunity, regulation of cell shape and migration, and mitogenic regulation (1-3). Rac1 and Rac2 participate in the regulation of ROS generation in several cell types (4, 5), especially in the neutrophil, where Rac2 provides one of several "triggers" for activation of the phagocyte respiratory burst oxidase, a superoxide-generating NADPH-oxidase that participates in host defense against invading microbes. In addition to regulation of ROS 3 production in phagocytes, there is growing evidence for Rac1 regulation of ROS generation in other cell types. For example, Ras-transformed fibroblasts overproduce superoxide, and ROS generation is inhibited by a dominant negative mutant form of Rac1 (6); also, stimuli that increase Rac1-GTP in gastric epithelial cells increase ROS production (7). Mutationally activated Rac1 induces ROS formation (5,7,8), and second site mutations showed that Rac1 activation of ROS production correlates with mitogenic stimulation, but not with actin polymerization or JNK activation by Rac1 (5). Rac1-regulated ROS production is also linked to neuronal differentiation (9), to growth and induction of cyclin D1 in airway smooth muscle (9), to shear stress-induced protein phosphorylations in vascular endothelium (10), and to platelet-derived growth factor-induced proliferation in vascular smooth muscle (11). Despite a clear association between Rac1 and ROS production in a variety of cells, the ROS-generating target(s) of Rac1 have not been convincingly elucidated, and the source has been specula...