Transepithelial transport of Na⁺ across the lung epithelium via amiloride-sensitive Na⁺ channels (ENaC) regulates fluid volume in the lung lumen. Activators of AMP-activated protein kinase (AMPK), the adenosine monophosphate mimetic AICAR and the biguanide metformin decreased amiloride-sensitive apical Na⁺ conductance (G_Na+) in human H441 airway epithelial cell monolayers. Cell-attached patch clamp recordings identified two distinct constitutively active cation channels in the apical membrane that were likely to contribute to G_Na+: a 5 pS highly Na⁺ selective ENaC-like channel (HSC) and a 18 pS non-selective cation channel (NSC). Substituting NaCl with NMDG-Cl in the patch pipette solution shifted the reversal potentials of HSC and NSC from +23 mV to -38 mV and 0 mV to -35 mV respectively. 1µM amiloride inhibited HSC activity and 56% of I_sc whereas 10µM amiloride partially reduced NSC activity and inhibited 30% of I_sc. Neither conductance was associated with CNG channels as there was no effect of 10 µM pimoside on I_sc, HSC or NSC activity and 8-bromo-cGMP (0.3-0.1 mM) did not induce or increase HSC or NSC activity. Pre-treatment of H441 monolayers with 2mM AICAR inhibited HSC and NSC activity by 90 % and this effect was reversed by the AMPK inhibitor Compound C. All three ENaC channel proteins were identified in the apical membrane of H441 monolayers but no change in their abundance was detected after treatment with AICAR. In conclusion, activation of AMPK with AICAR in H441 cell monolayers is associated with inhibition of two distinct ENaC-like amiloride-sensitive Na⁺-permeable channels by a mechanism that likely reduces channel open probability.