gp91phox (Nox2), the catalytic subunit of the superoxide-generating respiratory burst oxidase, is regulated by subunits p47phox and p67phox. Nox1, a homolog of gp91 phox, is regulated by NOXO1 and NOXA1, homologs of p47phox and p67phox, respectively. For both Nox1 and gp91phox, an organizer protein (NOXO1 or p47phox) cooperates with an activator protein (NOXA1 or p67phox) to regulate the catalytic subunit. Herein, we investigate the subunit regulation of Nox3 compared with that of other Nox enzymes. Nox3, like gp91phox was activated by p47phox plus p67phox. Whereas gp91phox activity required the protein kinase C activator pliorbol myristate acetate (PMA), Nox3 activity was already high without PMA, but was further stimulated ∼30% by PMA. gp91 phox was also activated by NOXO1/NOXA1 and required PMA for high activity. gp91phox regulation required an intact activation domain in the activator protein, as neither p67phox(V204A) nor NOXA1 (V205A) were effective. In contrast, p67phox(V204A) was effective (along with p47phox) in activating Nox3. Unexpectedly, Nox3 was strongly activated by NOXO1 in the absence of NOXA1 or p67phox. Nox3 activity was regulated by PMA only when p47phox but not NOXO1 was present, consistent with the phosphorylation-regulated autoinhibitory region in p47- phox but not in NOXO1. Deletion of the autoinhibitory region from p47phox rendered this subunit highly active in the absence of PMA toward both gp91phox and Nox3, and high activity required an activator subunit. The unique regulation of Nox3 supports a model in which multiple interactions with regulatory subunits stabilize an active conformation of the catalytic subunit.