We have examined the effect of ONOO- on a cloned epithelial sodium channel (αβγ-rENaC) expressed in Xenopus oocytes. We used 3-morpholinosydnonimine (SIN-1) to concurrently generate nitric oxide (·NO) and superoxide (O2+) which react at physiological pH to form ONOO-, a species known to promote protein nitration. Under control conditions oocytes displayed an amiloride-sensitive whole-cell conductance of 8.6±2.4 μS at -100 mV, When incubated with SIN-1 (1 mM) for 2 hr this conductance was reduced to 0.5±0.8 μS. To evaluate whether the observed inhibition was due to ONOO- or ·NO, we also exposed oocytes to SIN-1 in the presence of superoxide dismutase (SOD) which scavenges O2 leading to ·NO instead of ONOO-. Conductance values remained at control levels in the SOD cotreated oocytes. Moreover, when ONOO- was directly scavenged by adding urate, the effect of SIN-1 on the amiloride-sensitive conductance was also abolished. These data indicate that ONOO-, and not ·NO, significantly inhibits αβγ-rENaC function, thus supporting evidence that Na+ transport function is impaired in a variety of tissues upon exposure to this reactive species.