Although clusters of alveoli form the acinus, which is the most distal respiratory unit, it is not known whether inter-alveolar communication coordinates acinar surfactant secretion. To address this, we applied real-time digital imaging in conjunction with photo-excited Ca²⁺ uncaging in intact alveoli of the isolated, blood perfused rat lung. We loaded alveolar cells with the Ca²⁺ cage, o-nitrophenyl EGTA AM (NPEGTA-AM) together with the fluorophores, fluo 4, or LysoTracker Green (LTG) to determine respectively, the cytosolic Ca²⁺ concentration (Ca²⁺cyt), or type 2 cell secretion. To uncage Ca²⁺ from NPEGTA, we exposed a region in a selected alveolus to high-intensity UV illumination. As a result, fluo 4 fluorescence increased while LTG fluorescence decreased in the photo-targeted region, indicating that uncaging both increased Ca²⁺cyt and induced secretion. Concomitantly, Ca²⁺cyt increases conducted from the uncaging site induced type 2 cell secretion in both the selected alveolus as well as in neighboring alveoli, indicating the presence of inter-alveolar communication. These conducted responses were inhibited by pre-treating the alveoli with the connexin 43 (Cx43)-inhibiting peptides gap 26 and gap 27. However, although the conducted Ca²⁺cyt increase diminished with distance from the uncaging site, type 2 cell secretion rates were similar at all locations. We conclude that Cx43-dependent, inter-alveolar Ca²⁺ signals regulate type 2 cell secretion in adjacent alveoli. Such inter-alveolar communication might facilitate acinar coordination of alveolar function.