We examined the effect of peroxynitrite (ONOO-) on the cloned rat epithelial Na+ channel (αβγ-rENaC) expressed in Xenopus oocytes. 3- Morpholinosydnonimine (SIN-1) was used to concurrently generate nitric oxide (·NO) and superoxide (O2/-·), which react to form ONOO-, a species known to promote protein nitration and oxidation. Under control conditions, oocytes displayed an amiloride-sensitive whole cell conductance of 7.4 ± 2.8 (SE) μS. When incubated at 18°C with SIN-1 (1 mM) for 2 h (final ONOO- concentration = 10 μM), the amiloride-sensitive conductance was reduced to 0.8 ± 0.5 μS. To evaluate whether the observed inhibition was due to ONOO- , as opposed to ·NO, we also exposed oocytes to SIN-1 in the presence of urate (500 μM), a scavenger of ONOO- and superoxide dismutase, which scavenges O2/-· converting SIN-1 from an ONOO- to an ·NO donor. Under these conditions, conductance values remained at control levels following SIN-1 treatment. Tetranitromethane, an agent that oxidizes sulfhydryl groups at pH 6, also inhibited the amiloride-sensitive conductance. These data suggest that oxidation of critical sulfhydryl groups within rENaC by ONOO- directly inhibits channel activity.