Hypoxia inhibits transepithelial Na+ transport. However, the mechanism remains unresolved. We previously showed that pharmacological activation of adenosine monophosphate activated protein kinase (AMPK), inhibited amiloride-sensitive Na+ transport across H441 human airway epithelial cells. We have tested the hypothesis that hypoxia activates AMPK to decrease Na+ transport in the airway epithelium. Using high-performance liquid chromatography, we analysed the intracellular adenine nucleotide concentrations in the cell. We also examined the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) (a downstream target of AMPK). The effect of hypoxia on transepithelial Na+ transport was studied using Ussing chamber experiments. Compared to cells in normoxia (control, 21% O2), exposure to 0.2% O2 (for 1 hour) increased the AMP:ATP ratio from 0.006 ± 0.0004 to 0.030 ± 0.002 (p<0.01, n=3). Consistent with the notion that AMPK is activated by increased AMP:ATP, anoxia induced a 6.0 ± 0.7 (p<0.01, n=4) fold increase in phospho/total AMPK, compared to control. Phospho/total ACC was also increased by 2.2 ± 0.2 (p<0.01, n=4). Exposure to 3% O2 did not significantly affect the AMP:ATP ratio or the phosphorylation of AMPK and ACC. The cellular ATP level was not significantly affected by 3% or 0.2% O2. Exposure to 3% and 0.2% O2 reduced the amiloride-sensitive (10µM) short circuit current (Isc) from 20.4 ± 2.4 to 16.1 ± 1.9 µA/cm2 (p<0.03, n=3) and from 19.2 ± 1.2 to 10.1 ± 0.9 µA/cm2 (p<0.01, n=16) respectively compared to control. Ouabain-sensitive current was also reduced from 93.0 ± 4.7 to 72.7 ± 9.6 µA/cm2 (p<0.05, n=3) and from 76.6 ± 9.7 to 45.1 ± 5.1 µA/cm2 (p<0.01, n=16) respectively compared to control. Tin Protoporphyrin IX (SnPP), an inhibitor of haem oxygenase, did not prevent the inhibitory effect of hypoxia on amiloride- and ouabain-sensitive current. Reactive oxygen species (ROS) scavenger N-Acetyl-L-cysteine (NAC) (10 mM) decreased both amiloride (20.4 ± 2.4 to 10.0 ± 1.1 µA/cm2, p<0.01, n=3) and ouabain-sensitive current (92.9 ± 4.7 to 34.1 ± 5.8 µA/cm2, p<0.01, n=3) in control cells. Interestingly, exposure to 3% or 0.2 % O2, in the presence of NAC, induced a increase in amiloride (from 10.0 ± 1.1 to 16.1 ± 1.7 and 13.3 ± 1.2, p<0.05, n=3) and ouabain-sensitive current (from 34.1 ± 5.8 to 93.4 ± 2.6 and 83.3 ± 6.9, p<0.05, n=3). Exposure to 3% and 0.2% O2 inhibited transepithelial Na+ transport. However, 0.2% O2, which increased the AMP:ATP ratio and activated AMPK, produced a more potent inhibition. This indicates a potential role for AMPK in the regulation of transepithelial Na+ transport during hypoxia. We also speculate that ROS are involved in the regulation of Na+ transport by hypoxia. Supported by the BBSRC and St George’s University of London.