Activity of the Nox1 centered NADPH oxidase complex depends on the activator NoxA1 and the organizer NoxO1. NoxO1 itself is not involved in electron transfer for superoxide formation. Instead, by creating proximity of all components in the right place NoxO1 enables a constitutive formation of reactive oxygen species by that complex. All subunits should form the complex in a 1:1:1 ratio. When analyzing different cell lines in this study, we found an unequal expression of the component on mRNA level with an excess of NoxO1. Even with plasmid-based overexpression of individual components of Nox1 centered NADPH oxidase complex results in different expression of their mRNA, with NoxO1 mRNA being best expressed. Despite an unchanged high level of NoxO1 mRNA over a wide range of transfected plasmid, protein expression is increased with accelerating plasmid concentrations. We thought to analyze the ability of NoxO1 to induce ROS formation, when present in different ratio to Nox1 and NoxA1. To this end, we used Hek293 cells with constitutive expression of Nox1 and NoxA1 transfected with increasing concentrations of NoxO1. Our results suggest that ROS formation by the Nox1 centered NADPH oxidase strongly depends on the level of NoxO1 and a surplus of NoxO1 further increases the activity of the complex.