NADPH oxidase is the most important source of oxygen-derived radicals (ROS) in the vascular wall. In vascular smooth muscle cells (VSMC), NADPH oxidase is characterized by the expression of the membrane subunit Nox1, which is activated by cytoplasmic proteins binding to its activation domain. We set out to identify the cytoplasmic protein involved in NADPH oxidase activation in mouse VSMC. Western blot analysis revealed that human endothelial cells and leukocytes but not VSMC from the aorta of the rat and the mouse express the classic NADPH oxidase activator p67phox. In mouse VSMC, however, the p67phox homologue Noxa1 was detected. Using antibodies generated against mouse Noxa1, the protein was observed in the cytosolic fraction of mouse VSMC with a molecular weight of about 51 kDa. Immunohistochemistry revealed that Noxa1 is expressed in the smooth muscle layer but not in endothelium or the adventitia of the mouse carotid artery. Fluorescent fusion proteins of Noxa1 were observed to be expressed in the cytoplasm of VSMC and coexpression of the NADPH oxidase organizer Noxo1 targeted the complex to membrane. An antisense plasmid of Noxa1 attenuated the endogenous Noxa1 protein expression in VSMC. This plasmid attenuated the ROS formation in mouse VSMC as detected using L012 chemiluminescence and prevented the agonist-induced ROS production in response to basic fibroblast growth factor and epidermal growth factor. In conclusion, these data indicate that Noxa1 replaces p67phox in VSMC and plays a central role in the activation of the NADPH oxidase in the vascular wall.