NADPH oxidase-derived reactive oxygen species are important signalling molecules. Isoforms of the NADPH oxidase exist which differ in physiological function, mode of activation, activity and potentially the radical species generated. The homologue Nox1 contributes to agonist-signalling, whereas Nox4 is involved in long-term processes. We aimed to uncover differences in the molecular functions of Nox1 and Nox4 to allow understanding the differential physiological functions of the proteins. Nox1 and Nox4 show 36 % homology in the amino acid sequence. If transfected into HEK293 cells, Nox4 but not Nox1 predominantly localizes to the endoplasmic reticulum and is constitutively active. In intact cells, Nox1, if co-transfected with activator proteins, releases superoxide anions (SA) into the extracellular space, whereas Nox4 produces H2O2. In membrane fractions however Nox4 also generates SA. Chimeric proteins containing the transmembrane part of one isoform and the cytoplasmic tail of the other one were generated. The variant consistent of AA1-289 of Nox1 and AA304-578 of Nox4 is constitutively active whereas the variant AA1-303 / Nox4 and AA290-564 / Nox1 is inactive and could also not be activated by cytosolic subunits. Therefore, the constitutive activity of Nox4 is located in the cytoplasmic tail. The activation of Nox1 however requires more than the cytoplasmic tail, and further interactions with the transmembrane domains have to occur.