Insulin and aldosterone are major hormones controling sodium reabsorption in the distal nephron, hence controlling also extracellular fluid volume and blood pressure. We have demonstrated that insulin and aldosterone as well as hydrogen peroxide induce the generation of phosphatidylinositol 3,4,5-trisphosphate (PIP₃) and a rise in sodium transport. The aim of the present study was to investigate whether the stimulation of the PI 3-kinase pathway could result from local hydrogen peroxide production. We incubated A6 cell monolayers with the oxidation-sensitive fluorescent probe 5,6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H₂DCF-DA) under various experimental conditions. Insulin induced an immediate increase in fluorescence, indicative of ROS production that begins within two minutes after insulin stimulation was maximal at 5 min and lasted at least 30 min. Aldosterone induced an increase in fluorescence that begins 30 min after aldosterone addition, was maximal at 50 min and lasted for 24 hr. In both cases the increase in fluorescence was inhibited by a chelator of O₂^- (Nitro Blue Tetrazolium, 100 mM) and by a chelator of H₂O₂ (ebselen, 50 mM), These drugs also inhibited the rise in sodium transport induced by both hormones. Several inhibitors of the NADPH oxidase (Nox) enzymes (diphenyleneiodonium, phenylarsine oxide and plumbagin) also inhibited both the generation of ROS and the stimulation of sodium transport induced either by insulin or by aldosterone. In conclusion the present results support the hypothesis that activation of some Nox enzyme is responsible for increased hydrogen production, PI 3-kinase activation leading to increased sodium transport following exposure to either insulin or aldosterone.