In a previous work the involvement of a fenamate-sensitive Ca²⁺-activated nonselective cation channel (NSCC) in free radical-induced rat liver cell necrosis was demonstrated (5). Therefore, we studied 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). Single channel currents were recorded in HTC cells using the excised inside-out configuration of the patch-clamp technique. In this cell line, we characterize a 19 pS Ca²⁺-activated, ATP and fenamate- sensitive NSCC nearly equally permeable to monovalent cations. 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 (^.OH) scavenger MCI-186 inhibited the effect of H₂O₂, indicating that the increase in P_o was mediated by ^.OH. Exposure of the patch membrane to the oxidizing agent 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) had a similar effect to ^.OH. The increase in P_o induced by ^.OH or DTNB was not reverted by preventing ^.OH formation or by DTNB washout, respectively. However, the reducing agent dithiothreitol (DTT) completely reversed the effects on P_o of both 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. 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.