The integral membrane protein p22phox forms a heterodimeric enzyme complex with NADPH oxidases (Noxs) and is required for their catalytic activity. Nox4, a Nox linked to cardiovascular disease, angiogenesis, and insulin signaling, is unique in its ability to produce hydrogen peroxide constitutively. To date, p22phox constitutes the only identified regulatory component for Nox4 function. To delineate structural elements in p22phox essential for formation and localization of the Nox4-p22 phox complex and its enzymatic function, truncation and point mutagenesis was used. Human lung carcinoma cells served as a heterologous expression system, since this cell type is p22phox-deficient and promotes cell surface expression of the Nox4-p22phox heterodimer. Expression of p22phox truncation mutants indicates that the dual tryptophan motif contained in the N-terminal amino acids 6-11 is essential, whereas the C terminus (amino acids 130-195) is dispensable for Nox4 activity. Introduction of charged residues in domains predicted to be extracellular by topology modeling was mostly tolerated, whereas the exchange of amino acids in predicted membrane-spanning domains caused loss of function or showed distinct differences in p22phox interaction with various Noxs. For example, the substitution of tyrosine 121 with histidine in p22phox, which abolished Nox2 and Nox3 function in vivo, preserved Nox4 activity when expressed in lung cancer cells. Many of the examined p22phox mutations inhibiting Nox1 to -3 maturation did not alter Nox4-p22phox association, further accenting the differences between Noxs. These studies highlight the distinct interaction of the key regulatory p22phox subunit with Nox4, a feature which could provide the basis for selective inhibitor development.