We have used HyPer, a ratiometric GFP-based biosensor, to follow H2O2dynamics in live cells. We have found that activation of the EGF receptor in epithelial cells leads to sustained generation of intracellular H2O2, which is blocked by apocynin, an inhibitor of the plasma membrane NADPH oxidase assembly. Apocynin also blocked HeLa cell proliferation induced by EGF, indicating that NADPH oxidase is critically involved. However, apocynin failed to alter the kinetics of EGF-stimulated ERK1/2 activation. We conclude that NADPH oxidase and intracellular H2O2are important downstream targets of EGF receptor that mediate the proliferation response by mechanisms distinct from activation of the classical ERK1/2 MAP-kinase pathway. © 2010 Pleiades Publishing, Ltd.