We studied the effects of two mutations of the extracellular loop of the α-subunit of the (ENaC) on amiloride-sensitive current in Xenopus laevis oocytes and the inhibition of this current by 3-morpholinosydnonimine (SIN-1). Injection of oocytes with wild-type (wt) α-,β-,γ-rENaC cRNA (8.3 ng/subunit) resulted 48-72 h later in inward Na+ currents (-5.5 ± 0.8 μA; means ± SE at -100 mV; n = 21), which were completely inhibited by amiloride. Oocytes injected with either α Y279A- or αY283A- and β-,γ-rENaC cRNAs had significantly lower Na+ currents. Furthermore, α Y279A-,β-,γ-rENaC-injected oocytes had a higher K i for amiloride (0.54 ± 0.97 vs. 0.10 ± 0.04 μM; P < 0.01). Exposure of oocytes to SIN-1 (1 mM) for 5 min decreased both total Na+ and amiloride-sensitive currents across wt and α Y279A- but not αY2S3A-,β-,γ-rENaC. Furthermore, exposure to SIN-1 increased the Ki for amiloride across wt but not αY279A-,β-,γ-rENaC-injected oocytes. These data indicate that both tyrosines are important for proper ENaC function and their oxidative modifications contribute to altered ENaC function.