Extracellular ATP and other nucleotides are important autocrine/paracrine mediators that regulate diverse processes critical for lung function, including mucocilliary clearance, surfactant secretion and local blood flow. Cellular ATP release is mechanosensitive, however, the impact of physical stimuli on ATP release during breathing has never been tested in intact lungs in real-time and remains elusive. In this pilot study, we investigated inflation-induced ATP release in rat lungs ex-vivo by real-time luciferin-luciferase (LL) bioluminescence imaging coupled with simultaneous infrared tissue imaging to identify ATP-releasing sites. With LL solution introduced into airspaces brief inflation of such edematous lung (1-s, ~20 cmH2O) induced transient (<30s) ATP release in a limited number of air-inflated alveolar sacs during their recruitment/opening. Released ATP reached concentrations of ~10-6 M, relevant for autocrine/paracrine signaling but it remained spatially restricted to single alveolar sacs or their clusters. ATP release was stimulus-dependent, prolonged (100-s) inflation evoked long-lasting ATP release which terminated upon alveoli deflation/de-recruitment while cyclic inflation/suction produced cyclic ATP release. With LL introduced into blood vessels, inflation induced transient ATP release in many small patch-like areas the size of alveolar sacs. Findings suggest that inflation induces ATP release in both alveoli and the surrounding blood capillary network; the functional units of ATP release presumably consist of alveolar sacs or their clusters. Our study demonstrates the feasibility of real-time ATP-release imaging in ex-vivo lungs and provides the first direct evidence of inflation-induced ATP release in lung airspaces and in pulmonary blood capillaries, highlighting the importance of purinergic signaling in lung function.