Endothelial cells (EC) express constitutively two major isoforms (Nox2 and Nox4) of the catalytic subunit of NADPH oxidase, which is a major source of endothelial reactive oxygen species. However, the individual roles of these Noxes in endothelial function remain unclear. We have investigated the role of Nox2 in nutrient deprivation-induced cell cycle arrest and apoptosis. In proliferating human dermal microvascular EC, Nox2 mRNA expression was low relative to Nox4 (Nox2:Nox4 approximately 1:13), but was upregulated 24 h after starvation and increased to 8+/-3.5-fold at 36 h of starvation. Accompanying the upregulation of Nox2, there was a 2.28+/-0.18-fold increase in O2.- production, a dramatic induction of p21cip1 and p53, cell cycle arrest, and the onset of apoptosis (all p<0.05). All these changes were inhibited significantly by in vitro deletion of Nox2 expression and in coronary microvascular EC isolated from Nox2 knockout mice. In Nox2 knockout cells, although there was a 3.8+/-0.5-fold increase in Nox4 mRNA expression after 36 h of starvation (p<0.01), neither O2.- production nor the p21cip1 or p53 expression was increased significantly and only 0.46% of cells were apoptotic. In conclusion, Nox2-derived O2.-, through the modulation of p21cip1 and p53 expression, participates in endothelial cell cycle regulation and apoptosis.