The stress-activated metabolic sensor AMP-activated protein kinase (AMPK) inhibits both the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial Na+ channel (ENaC) in polarized epithelial cell lines (1-4). In addition, AMPK subcellular localization is disrupted and AMPK activity is up-regulated in primary polarized cystic fibrosis (CF) human bronchial epithelial (HBE) cells relative to non-CF HBE cells, an effect that may help tamp down the secretion of pro-inflammatory cytokines (5). We have tested in primary CF versus non-CF HBE cells the effects of the mechanistically distinct pharmacological AMPK activators metformin and 5-aminoimidazole-4-carboxamide-1-β-D-riboside (AICAR) on various functionally relevant parameters that contribute to CF lung disease: ENaC and CFTR-dependent short-circuit currents (Isc), airway surface liquid (ASL) reabsorption rates, and pro-inflammatory cytokine secretion (6). As compared with controls, AMPK activation following overnight treatment with either metformin (2-5 mM) or AICAR (1 mM) substantially inhibited ENaC-dependent Isc in both cell types as well as CFTR-dependent Isc in non-CF cells. ASL reabsorption rates over time were measured by live-cell confocal microscopy following apical addition of PBS containing Texas Red-dextran. Measured ASL heights were significantly greater following AICAR and metformin treatment at 60 min in both CF and non-CF cells relative to controls, suggesting that AMPK-dependent inhibition of ENaC in airway cells slows apical fluid reabsorption. Extending our previous results obtained using immortalized CF versus non-CF cell lines, we also found that both metformin and AICAR decreased the apical secretion of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin 6, and interleukin 8 in primary CF and non-CF HBE cells, both in the presence and absence of prior stimulation with lipopolysaccharide (LPS). Finally, much lower concentrations of metformin (0.03 – 1 mM) given over longer time periods, corresponding more closely to drug levels achieved therapeutically in vivo, were sufficient to cause inhibitions of ENaC-dependent currents and pro-inflammatory cytokine levels in CF HBE cells. The inhibition of ENaC and pro-inflammatory cytokine secretion by AMPK may be mediated in part by AMPK-dependent inhibition of IκB kinase-β, an upstream regulator of the NF-κB pathway. These findings suggest that novel therapies to activate AMPK in the CF airway may be of benefit both by blunting excessively high ENaC activity and thus ASL hyperabsorption and by reducing excessive airway inflammation, which are both major contributors to CF lung disease.