Oxygen radicals play an important role in signal transduction and have been shown to influence epithelial sodium channel (ENaC) activity. We show that aldosterone, the principal hormone regulating renal ENaC activity, increases superoxide (O₂⁻) production in A6 distal nephron cells. Aldosterone (50 nM to 1.5 μM) induced increases in dihydroethidium fluorescence in a dose-dependent manner in confluent A6 epithelial cells. Using single-channel measurements, we showed that sequestering endogenous O₂⁻ (with the O₂⁻ scavenger 2,2,6,6-tetramethylpiperidine 1-oxyl) significantly decreased ENaC open probability from 0.10 ± 0.03 to 0.03 ± 0.01. We also found that increasing endogenous O₂⁻ in A6 cells, by applying a superoxide dismutase inhibitor, prevented nitric oxide (NO) inhibition of ENaC activity. ENaC open probability values did not significantly change from control values (0.23 ± 0.05) after superoxide dismutase and 1.5 μM NO coincubation (0.21 ± 0.04). We report that xanthine oxidase and hypoxanthine compounds increase local concentrations of O₂⁻ by ∼30%; with this mix, an increase in ENaC number of channels times the open probability (from 0.1 to 0.3) can be achieved in a cell-attached patch. Our data also suggest that O₂⁻ alters NO activity in a cGMP-independent mechanism, since pretreating A6 cells with ODQ compound (a selective inhibitor of NO-sensitive guanylyl cyclase) failed to block 2,2,6,6-tetramethylpiperidine 1-oxyl inhibition of ENaC activity.