Intracellular accumulation of ROS, such as hydrogen peroxide, superoxide anion and hydroxyl radical results from normal metabolic processes or toxic insults. The degree of oxidative stress is determined by the balance between free radical synthesis and degradation. Alterations of this balance have been associated to aging and several pathological processes such as inflammation and ischemia-reperfusion. High, long-standing levels of ROS can cause necrosis and direct treatment of cells with oxidants has been demonstrated to induce necrosis, which appears to be the result of acute cellular dysfunction in response to severe stress conditions or after exposure to toxic agents. Intracellular calcium and sodium overload has been recognised as a critical step in necrosis. Previously, the involvement of a fenamate-sensitive Ca²⁺-activated nonselective cation channel (NSCC) in free radical-induced rat liver cell necrosis was demonstrated (1). Based on that observation, the effect of radical oxygen species (ROS) and oxidizing agents on the gating behavior of a NSCC in a liver-derived epithelial cell line (HTC) has been now addressed. Single channel currents were recorded in HTC cells using the excised inside-out configuration of the patch-clamp technique. In this cell line, a 19 pS Ca²⁺-activated, ATP- and fenamate-sensitive NSCC nearly equally permeable to monovalent cations is characterised. In the presence of Fe²⁺, exposure of the intracellular side of NSCC to H₂O₂ increased their open probability (Po) by ∽40% without affecting the unitary conductance. Desferroxamine as well as the hydroxyl radicals (null;OH) scavenger MCI-186 inhibited the effect of H₂O₂, indicating that the increase in Po was mediated by null;OH. Exposure of the patch membrane to the oxidizing agent 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB) had a similar effect to null;OH. The increase in Po induced by null;OH or DTNB was not reverted by preventing null;OH formation or by DTNB washout, respectively. However, the reducing agent dithiothreitol (DTT) completely reversed the effects on Po of both null;OH and DTNB. A similar increase in Po was observed by applying the physiological oxidizing molecule GSSG. Moreover, GSSG-oxidized channels showed enhanced sensitivity to Ca²⁺. The effect of GSSG was fully reversed by GSH (2). These results suggest an intracellular site(s) of action of oxidizing agents on cysteine targets on the fenamate-sensitive NSCC protein implicated in epithelial cell necrosis.