Current theories of CF pathogenesis predict different predisposing “local environmental” conditions and sites of bacterial infection within CF airways. Here we show that, in CF patients with established lung disease, Psuedomonas aeruginosa was located within hypoxic mucopurulent masses in airway lumens. In vitro studies revealed that CF-specific increases in epithelial O₂ consumption, linked to increased airway surface liquid (ASL) volume absorption and mucus stasis, generated steep hypoxic gradients within thickened mucus on CF epithelial surfaces prior to infection. Motile P. aeruginosa deposited on CF airway surfaces penetrated into hypoxic mucus zones and responded to this environment with increased alginate production. With P. aeruginosa growth in oxygen restricted environments, local hypoxia was exacerbated and frank anaerobiosis, as detected in vivo, resulted. These studies indicate that novel therapies for CF include removal of hypoxic mucus plaques and antibiotics effective against P. aeruginosa adapted to anaerobic environments.