Insulin-stimulated sodium transport across A6 cell (derived from amphibian distal nephron) monolayers involves the activation of a PI 3-kinase. We previously demonstrated that exogenous addition of H₂O₂ to the incubation medium of A6 cell monolayers provokes an increase in PI 3-kinase activity and a subsequent rise in sodium transport. We therefore questioned whether insulin would produce an intracellular burst of H₂O₂ leading to PI 3-kinase activation and subsequent increase in sodium transport. An acute production of reactive oxygen species (ROS) in A6 cells incubated with the oxidation-sensitive fluorescent probe (5,6-chloromethyl-2,7-dichlorodihydrofluorescein diacetate) was already detected after two minutes of insulin stimulation. This fluorescent signal and the increase in sodium transport were completely inhibited in monolayers incubated with peggylated catalase indicating that H₂O₂ is the main intracellular ROS produced upon insulin stimulation. Likewise, preincubation of monolayers with different chelators of either superoxide (O₂^.-) (Nitro Blue Tetrazolium, 100 µM) or H₂O₂ (Ebselen, 50 µM), or blockers of NADPH oxidase (Nox) enzymes (diphenyleneiodonium, 5 µM; phenylarsine oxide, 1 µM and Plumbagin, 30 µM) prevented both insulin-stimulated H₂O₂ production and insulin-stimulated sodium transport. Furthermore, diphenyleneiodonium pretreatment inhibited the recruitment of p85 PI 3-kinase regulatory subunit in an anti-phosphotyrosine immunoprecipitate in insulin stimulated cells. In contrast, PI-103, an inhibitor of class IA PI 3-kinase inhibited insulin-stimulated sodium transport but did not significantly reduce insulin-stimulated H₂O₂ production. Taken together, our data suggest that insulin induces an acute burst of H₂O₂ production which participates in an increase in PIP₃ production and subsequently a stimulation of the sodium transport.