Reactive oxygen species (ROS) are important signal transduction molecules in ligand-induced signaling, regulation of cell growth, differentiation, apoptosis and motility. Recently NADPH oxidases (Nox) homologous to Nox2 (gp91^phox) of phagocyte cytochrome b₅₅₈ have been identified, which are an enzymatic source for ROS generation in epithelial cells. This study was undertaken to delineate the requirements for ROS generation by Nox4. Nox4, in contrast to other Nox proteins, produces large amounts of hydrogen peroxide constitutively. Known cytosolic oxidase proteins or the GTPase Rac are not required for this activity. Nox4 associates with the protein p22^phox on internal membranes, where ROS generation occurs. Knockdown and gene transfection studies confirmed that Nox4 requires p22^phox for ROS generation. Mutational analysis revealed structural requirements affecting expression of the p22^phox protein and Nox activity. Mechanistic insight into ROS regulation is significant for understanding fundamental cell biology and pathophysiological conditions.