Reactive oxygen species (ROS), which include superoxide, hydrogen peroxide and peroxynitrite, are generated in a deliberate and highly controlled fashion by virtually all eukaryotic cells to regulate the activity of redox-sensitive effector proteins such as tyrosine kinases and phosphatases, serine threonine kinases and nuclear transcription factors. However, in cardiovascular diseases such as hypertension, hypercholesterolemia and diabetes, ROS production in the artery wall is markedly elevated, leading to oxidative damage to cellular macromolecules, endothelial dysfunction, and inflammation, which are precursors for the development of atherosclerotic lesions. Yet, despite the wealth of evidence implicating ROS as important contributors to vascular disease, clinical trials assessing the effects of antioxidants (i.e. ROS scavengers) on cardiovascular events and progression of atherosclerosis have been disappointing. Thus, identification of the enzyme(s) responsible for excessive ROS production during vascular pathophysiology may lead to new therapies that more effectively reduce ROS levels in the vascular wall by blocking their production at the source. NADPH oxidases are a family of ROS-generating enzymes comprising a membrane-bound catalytic subunit (Nox1-5), a smaller protein that stabilises the Nox subunit within membranes (p22phox), and up to three cytosolic regulatory subunits including an organiser protein (p47phox or Noxo1), an activator protein (p67phox or Noxa1) and a GTPase (Rac1/2). At least three isoforms of NADPH oxidase are expressed in the blood vessel wall, including Nox1-, Nox2- and Nox4-containing enzymes. We demonstrated that atherogenesis in apolipoprotein E-deficient (ApoE /-) mice is associated with marked increases in vascular ROS production and expression of Nox1 and Nox2, but downregulation of Nox4. Importantly the absence of Nox1 or Nox2 in novel Nox1-/-/ApoE-/- and Nox2-/-/ApoE-/- double-knockout mice, respectively, reduced vascular ROS production, increased NO bioavailability and limited atherosclerotic plaque development along the aorta, highlighting these two isoforms of NADPH oxidase as potential therapeutic targets. Thus, following our observation that the Nox substrate, NADPH, is an agonist at purinoceptors (Judkins et al., 2006), we demonstrated that purinoceptor antagonists, and several structurally related molecules lacking activity at purinoceptors, are conversely novel inhibitors of Nox activity. One of these (designated RADX092) is not only a powerful inhibitor of Nox2 activity in vitro (IC50~10µM), but is devoid of non-specific ROS scavenging activity, and of inhibitory effects against other NAD(P)H-utilizing enzymes such as NO synthase and xanthine oxidase. Moreover, it is orally active and reverses angiotensin II-induced hypertension in mice. Thus, we have not only established a cause and effect relationship for NADPH oxidase in atherosclerosis, but have provided proof-of-principal that small molecule inhibitors of these enzymes may represent promising therapeutics for vascular disease.