We have previously shown that activation of AMP-activated protein kinase (AMPK) with phenformin or AICAR decreased transepithelial amiloride-sensitive Na⁺ transport (I_amiloride), apical Na⁺ conductance (G_Na+) and ouabain-sensitive Na⁺-K⁺-ATPase (I_ouabain) activity in H441 lung epithelial cells (1). The biguanide, phenformin, is thought to activate AMPK by inhibition of Complex I of the mitochondrial respiratory chain and increasing the intracellular AMP:ATP ratio (2,3). In contrast, the adenosine analogue 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) activates AMPK by mimicking the effect of AMP without affecting cellular ATP levels in intact cells. By measuring short circuit current (I_sc) across H441 cell monolayers, we have further explored the effect of these drugs and the activation of AMPK on ion transport processes across H441 cells. Phenformin (5nM) and AICAR (2mM) evoked similar ~2.0-fold increase in AMPK activity in H441 cells from 0.03 ± 0.002 to 0.05 ± 0.008 and 0.04 ± 0.004 to 0.07 ± 0.005 nmols/min/mg, respectively. Activity was significantly increased at 30 min (p < 0.01, n = 3, respectively) and was maintained over 24 hours. Phenformin and AICAR inhibited transepithelial I_amiloride to minimal levels (~20% of control) at 4–8 hours with similar efficacy (t_1/2 32.5 min and 33.5 min, respectively, n = 3). In contrast, 5 mM phenformin maximally inhibited I_ouabain to 25% of control levels with a t_1/2 of 12 min (n = 3) but AICAR only inhibited I_ouabain to 66% of control levels with a t_1/2 of 58 min. Measurement of NADPH autofluorecence by excitation at 405 nm wavelength (indicative of inhibition of mitochondrial function) showed that phenformin but not AICAR was associated with increased NADPH fluoresecence (35.6 ± 0.7 fluorecence units, p < 0.01, n = 20). Transepithelial amiloride-sensitive Na⁺ transport is regulated by apical Na⁺ entry through amiloride-sensitive Na⁺ channels and extrusion via Na⁺-K⁺-ATPase. That phenformin and AICAR had similar effects on AMPK activity and I_amiloride but AICAR had a greater effect on I_amiloride than I_ouabain indicate that activation of AMPK inhibits Na⁺ entry as the rate-limiting step in transepithelial Na⁺ transport. Furthermore, the more potent effect of phenformin compared to AICAR on I_ouabain indicates that factors additional to AMPK activation, which could include ATP availability, modulate Na⁺K⁺ATPase activity in H441 lung epithelial cells.