Evidence show that diverse growth factors (GF) induce an increase of reactive oxygen species (ROS) (1) suggested to participate in cell proliferation. One putative role of these ROS is to modify the phosphorylation state of GF receptors by reversible inhibition of proteins tyrosine phosphatases. It has been also demonstrated that subtoxic oxidative challenges induce cell proliferation. On the other hand, a crucial role of Cl^– channels in cell proliferation has been suggested in different cells lines (2). Recently, we have demonstrated that hydrogen peroxide derived from NAD(P)H oxidase (NOX) activation plays an important role in the activation of volume-sensitive outward rectifying (VSOR) Cl^– channels (3). Therefore, we explored the possibility that intracellular ROS produced by epidermal growth factor (EGF) could be part of the mechanism leading to the activation of Cl^– currents implicated in cell proliferation (2). Acute exposure of HeLa cells to EGF (500 ng/ml) activated a Cl^– current with similar properties to VSOR Cl^– channels. This current was inhibited (> 90%) by intracellular application of DTT (5 mM). To examine whether EGF-induced increase in ROS was dependent on NOX activation, we tested a NOX inhibitor (DPI, 10 μM). Cells pretreated with DPI showed a significant inhibition (50 – 75%) of EGF-induced Cl^– current. Because DPI is a nonspecific blocker of NOX, inhibition of the EGF-induced Cl^– current by this compound should be interpreted with caution. To address this point more directly, HeLa cells were transiently transfected with a dominant negative mutant (p47^S379A) of the p47 subunit of NOX. This mutant is unable to translocate to the membrane, resulting in a loss of oxidase activity. As expected, the dominant negative mutant showed a significant reduction (> 80%) in the EGF-induced Cl^– current. In conclusion, our results indicate that EGF-induced chloride currents are dependent on ROS. Furthermore, the results presented here suggest that ROS generation induced by EGF is mediated by the activation of the oxidase subunit p47 component of the NAD(P)H oxidase.