Oxygen radicals play an important role in signal transduction and have been shown to influence epithelial sodium channel activity (ENaC). We show that aldosterone, the principal hormone regulating renal ENaC activity, increases superoxide (O₂^-) production in A6 distal nephron cells. 50nM-1.5µM aldosterone induced increases in DHE 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 TEMPO) significantly decreased ENaC open probability (Po) 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 Po values did not significantly change from control values (0.23±0.05) after superoxide dismutase and 1.5µM nitric oxide co-incubation (0.21±0.04). We report that xanthine oxidase and hypoxanthine compounds increase local concentrations of O₂^- by approximately 30%; with this mix an increase in ENaC NPo (from 0.1 to 0.3) can be achieved in a cell attached patch. Our data also suggests that O₂^- alters NO activity in a cGMP-independent mechanism, since pre-treating A6 cells with ODQ compound (a selective inhibitor of nitric oxide-sensitive guanylyl cyclase) failed to block TEMPO inhibition of ENaC activity.