Although its mode of action is still elusive, apocynin (APO) is by far the most popular NADPH oxidase inhibitor. We determined the mode of action of APO. In leukocytes, APO blocked the fMLP- and zymosan- but not the PMA-induced production of reactive oxygen species (ROS) . In HEK293 cells overexpressing NADPH oxidase isoforms, APO in contrast failed to inhibit the oxidase. It was however observed that the inhibitory effects of APO on ROS level in non-phagocytic cells are a consequence of a pronounced antioxidative effect of the compound. The scavenging properties of APO were most prominent for superoxide- generating systems coupled to peroxidase-dependent detector systems (such as luminol/HRP). Accordingly, the activation of redox-sensitive kinases such as p38 MAP-kinase and ERK1/2 in response to H2O2 or the redox cycler menadione were attenuated by APO, demonstrating that the compound acts as an antioxidant in live cells. In leukocytes, APO blocked the zymosan- as well as fMLP-mediated phosphorylation of Akt which is required for the process of NADPH oxidase assembly. In conclusion: Our observations demonstrate that APO is a powerful antioxidant and that the inhibition of the ROS formation in leukocytes is a consequence of an impaired activation rather then a direct inhibition of the NADPH oxidase.