Most mammalian cells types respond to cell swelling by activating a Cl^– current termed I_Cl,vol. Although this current has been well studied (1), the physical stimuli and the signal transduction pathways connecting cell swelling and the channels underlying I_Cl,vol are still elusive. In cells from a rat liver-derived cell line (HTC), it has been proposed that phosporylation of PLCγ is fundamental for the regulatory volume decrease (RVD) response (2), however, the upstream molecules and the role of these intracellular signalling components on the activation of I_Cl,vol are not known. Recent evidence indicate that reactive oxygen species may function as intracellular messengers leading to a reversible inactivation of some protein tyrosine phosphatases (PTPs) (3). In agreement, several authors demonstrate an increase in the phosphorylation state of PLCγ after exogenous application of hydrogen peroxide (H₂O₂) (4,5). In this work we show that a reduction of extracellular osmolarity (30% hypotonicity) increases the rate of production of intracellular H₂O₂ 15-fold. This increase was partially blocked (70 ± 11%) by pre-treatment of the cells with a non-specific inhibitor of NAD(P)H oxidase (diphenylene iodonium, DPI). To establish a correlation between the increase of H₂O₂ and the activation of I_Cl,vol, whole-cell chloride currents were studied using the nystatin perforated patch configuration. HTC cells exposed to hypotonicity developed a Cl^– current similar to that observed in other cell lines, however, pre-treatment with DPI inhibited this current by 77 ± 2%. Over-expression of a NAD(P)H oxidase dominant negative (p47^S379A) also abolished the development of this current, indicating a central role of this complex in the activation of I_Cl,vol. Extracellular application of H₂O₂ (20-200 μM) in isotonicity reversibly activated a chloride current that resembled I_Cl,vol. Consequently, internal application of DTT completely abolished the activation of this current. Application of a specific inhibitor of PLC, U73122 completely abolished the development of the current activated by hypotonicity or H₂O₂ (inhibition of 94 ± 1% and 93 ± 1%, respectively) suggesting that hydrogen peroxide may act upstream to PLC. These results indicate that hydrogen peroxide plays a central role in the activation of I_Cl,vol in a PLC-dependent manner.