BACKGROUND AND PURPOSE: Oxidative stress [i.e. increased levels of reactive 
oxygen species (ROS)] has been suggested as a pathomechanism of different 
diseases, although the disease-relevant sources of ROS remain to be identified. 
One of these sources may be NADPH oxidases. However, due to increasing concerns 
about the specificity of the compounds commonly used as NADPH oxidase 
inhibitors, data obtained with these compounds may have to be re-interpreted.
EXPERIMENTAL APPROACH: We compared the pharmacological profiles of the commonly 
used NADPH oxidase inhibitors, diphenylene iodonium (DPI), apocynin and 
4-(2-amino-ethyl)-benzolsulphonyl-fluoride (AEBSF), as well as the novel 
triazolo pyrimidine VAS3947. We used several assays for detecting cellular and 
tissue ROS, as none of them is specific and artefact free.
KEY RESULTS: DPI abolished NADPH oxidase-mediated ROS formation, but also 
inhibited other flavo-enzymes such as NO synthase (NOS) and xanthine oxidase 
(XOD). Apocynin interfered with ROS detection and varied considerably in 
efficacy and potency, as did AEBSF. Conversely, the novel NADPH oxidase 
inhibitor, VAS3947, consistently inhibited NADPH oxidase activity in low 
micromolar concentrations, and interfered neither with ROS detection nor with 
XOD or eNOS activities. VAS3947 attenuated ROS formation in aortas of 
spontaneously hypertensive rats (SHRs), where NOS or XOD inhibitors were without 
effect.
CONCLUSIONS AND IMPLICATIONS: Our data suggest that triazolo pyrimidines such as 
VAS3947 are specific NADPH oxidase inhibitors, while DPI and apocynin can no 
longer be recommended. Based on the effects of VAS3947, NADPH oxidases appear to 
be a major source of ROS in aortas of SHRs.