ATP is a paracrine regulator of critical airway epithelial cell functions but the mechanism of its release is poorly understood. Pannexins, proteins related to invertebrate innexins, form channels (called pannexons) that are able to release ATP from several cell types. Thus, ATP release via pannexons was examined in airway epithelial cells. Quantitative RT-PCR showed pannexin 1 expression in normal human airway epithelial cells during re-differentiation at the air-liquid interface (ALI), at a level comparable to alveolar macrophages. Pannexin 3 was not expressed. Immunohistochemistry showed pannexin 1 expression at the apical pole of airway epithelia. ALI cultures exposed to hypotonic stress released ATP to an estimated maximum of 255 ± 64 nM within 1 minute after challenge (n = 6 cultures from 3 different lungs) or to ~ 1.5 ± 0.4 µM, recalculated to a normal airway surface liquid volume. Using date and culture-matched cells (each n 16 from 4 different lungs), the pannexon inhibitors carbenoxolone (10 µM) and probenecid (1 mM) but not the connexon inhibitor flufenamic acid (100 µM) inhibited ATP release by > 60%. The drugs affected pannexin 1 currents in Xenopus oocytes expressing exogenous pannexin 1 correspondingly. In addition, suppression of pannexin 1 expression using lentivirus-mediated production of shRNA in differentiated airway epithelial cells inhibited ATP release upon hypotonic stress by ~60% as well. These data not only show that pannexin 1 is expressed apically in differentiated airway epithelial cells but also that it contributes to ATP release in these cells.