Background and purpose: AMP-activated protein kinase (AMPK) is activated by metformin, phenformin, and the AMP mimetic, 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside (AICAR). We have completed an extensive study of the pharmacological effects of these drugs on AMPK activation, adenine nucleotide concentration, transepithelial amiloridesensitive (I amiloride ) and ouabain-sensitive basolateral (I ouabain ) short circuit current in H441 lung epithelial cells. Experimental approach: H441 cells were grown on permeable filters at air interface. I amiloride , I ouabain and transepithelial resistance were measured in Ussing chambers. AMPK activity was measured as the amount of radiolabelled phosphate transferred to the SAMS peptide. Adenine nucleotide concentration was analysed by reverse phase HPLC and NAD(P)H autofluorescence was measured using confocal microscopy. Key results: Phenformin, AICAR and metformin increased AMPK (a1) activity and decreased I amiloride . The AMPK inhibitor Compound C prevented the action of metformin and AICAR but not phenformin. Phenformin and AICAR decreased I ouabain across H441 monolayers and decreased monolayer resistance. The decrease in I amiloride was closely related to I ouabain with phenformin, but not in AICAR treated monolayers. Metformin and phenformin increased the cellular AMP:ATP ratio but only phenformin and AICAR decreased cellular ATP. Conclusions and implications: Activation of a1-AMPK is associated with inhibition of apical amiloride-sensitive Na þ channels (ENaC), which has important implications for the clinical use of metformin. Additional pharmacological effects evoked by AICAR and phenformin on I ouabain, with potential secondary effects on apical Na þ conductance, ENaC activity and monolayer resistance, have important consequences for their use as pharmacological activators of AMPK in cell systems where Na þ K þ ATPase is an important component.