The purpose of this study was to investigate the direct effect of NH₃/NH on mouse epithelial Na⁺ channels (mENaC) expressed in Xenopus oocytes. Two-electrode voltage-clamp and ion-selective microelectrodes were used to measure the Na⁺ current, intracellular pH (pH_i), and ion activities in oocytes expressing mENaC. In oocytes expressing mENaC, removal of external Na⁺ reversibly hyperpolarized membrane potential by 129 ± 5.3 mV in the absence of 20 mM NH₄Cl but only by 100 ± 7.8 mV in its presence. Amiloride completely inhibited the changes in membrane potential. In oocytes expressing mENaC, butyrate (20 mM) caused a decrease in pH_i (0.43 ± 0.07) similar to the NH₄Cl-induced pH_i decrease (0.47 ± 0.12). Removal of Na⁺ in the presence of butyrate caused hyperpolarization that was not significantly different from that in the absence of butyrate at high pH_i (in the absence of NH₄Cl). Removal of external Na⁺ resulted in an outward current of 3.7 ± 0.8 µA (at 60 mV). The magnitude of this change in current was only 2.7 ± 0.7 µA when Na⁺ was removed in the presence of NH₄Cl. In oocytes expressing mENaC, NH₄Cl also caused a decrease in whole cell conductance at negative potential and an outward current at positive potential. In the presence of amiloride, steady-state current and the change in current caused by removal of Na⁺ were not different from zero. These results indicate that NH₄Cl inhibits Na⁺ transport when mENaC is expressed in oocytes. The inhibition of voltage changes is not due to intracellular acidification caused by NH₄Cl. Permeability and selectivity of ENaC to NH may play a role.