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Abstract: The superoxide-producing phagocyte NADPH oxidase gp91 phox /Nox2 and the non-phagocytic oxidases Nox1 and Nox3 each form a complex in the membrane with p22 phox , which provides both stabilization and a docking site for organizer proteins. The p22 phox -complexed Nox2 and Nox1 are dormant on their own, and their activation requires soluble supportive proteins such as a Nox organizer (p47 phox or Noxo1) and a Nox activator (p67 phox or Noxa1). The small GTPase Rac directly binds to the activators, and thus plays an essential role in the Nox2-based oxidase containing p47 phox and p67 phox or a positive role in Nox1 activity supported by Noxo1 and Noxa1. Although Nox3 complexed with p22 phox constitutively produce superoxide, the production can be enhanced by supportive proteins. Here we compare the roles of Rac in these p22 phox -dependent oxidases using the organizer and activator in different combinations. Expression of constitutively active Rac1(Q61L) is essential for activation of the Nox2- or Nox1-based oxidase containing the organizer p47 phox and either p67 phox or Noxa1. When these oxidases use Noxo1 as an organizer instead of p47 phox , they produce a small but significant amount of superoxide without expression of Rac1(Q61L), although the production is enhanced by Rac1(Q61L). Thus p47 phox is likely related to strict dependence on Rac. The Nox3-based oxidase has a similar tendency in the change of the dependence: Rac plays a positive role in Nox3 activation in the presence of p47 phox and either p67 phox or Noxa1, whereas Rac fails to upregulate Nox3 activity when p47 phox is replaced with Noxo1. We also demonstrate that, in the Nox3-based oxidase containing solely p67 phox as supportive protein, expression of Rac1(Q61L) enhances not only superoxide production but also membrane translocation of p67 phox . Since the enhancements are not observed with a mutant p67 phox defective in binding to Rac, this GTPase appear to directly recruit p67 phox to the membrane. [Copyright &y& Elsevier]