NADPH oxidases of the Nox family are considered important sources of cellular reactive oxygen species (ROS) production. This conclusion is in part based on the ability of NADPH to elicit a chemiluminescence signal in tissue/cell homogenates or membrane preparations in the presence of enhancers such as lucigenin, luminol or L012. However, the ability of these particular assays to specifically detect Nox activity and Nox-derived ROS has not been proven. We here demonstrate that combined knockout of the three main Nox enzymes of the mouse (Nox1-Nox2-Nox4 triple knockout) had no impact on NADPH-stimulated chemiluminescence signals in aorta, heart and kidney homogenates. In the NADPH-stimulated membrane assays, no effect of in vivo AngiotensinII pretreatment or deletion of Nox enzymes was observed. In in vitro studies in HEK293 cells, the overexpression of Nox5 or Nox4 markedly increased ROS production in intact cells whereas overexpression of Nox5 or Nox4 had no influence on the signal in membrane assays. In contrast, overexpression of nitric oxide synthase or cytochrome P450 enzymes resulted in an increased chemiluminescence signal in isolated membranes. On the basis of these observations we propose the hypothesis that NADPH-stimulated chemiluminescence-based membrane assays, as currently used, do not reflect Nox activity.