In airway epithelia, extracellular ATP regulates mucociliary clearance by stimulating cell surface P2Y₂ receptors. However, the mechanisms of ATP release and the magnitude of endogenous ATP accumulation in the thin layer of liquid covering airway surfaces are poorly understood. We monitored in real-time apical ATP concentrations adjacent to the cell surface of airway epithelial cells cultured in an air-liquid interface system. Firefly luciferase fused to the IgG-binding domain of Staphylococcus aureus protein A (SPA-luc) was bound to the apical surface of polarized airway epithelial Calu-3 cells via an antibody against an endogenous cell surface glycoprotein, i.e. MUC1. Basal ATP levels monitored by cell-attached luciferase were similar to those measured in bulk mucosal medium (depth 2.6 mm) (7.6±0.7 nM and 7.3±0.6 nM by cell-attached and bulk measurement, respectively, n=6). A robust ATP release was observed following a 33% hypotonic challenge by cell-attached luciferase, which was 20 times higher than the peak ATP level measured in bulk mucosal medium following a hypotonic challenge (645.4±63.5 nM and 34.1±5.6 nM by cell-attached and bulk measurement, respectively, n=6). Utilizing luciferase dissolved in a thin film covering the apical cell surface (height 12 μm), ATP levels were similar to those detected by cell-surface attached SPA-luc both in basal and stimulated conditions (7.1±0.6 nM and 614.1±56.2 nM in basal and stimulated conditions, respectively, n=6). Utilizing these methods, Calu-3 cells were tested to address whether cell-swelling induced ATP release from airway epithelia is dependent on changes in intracellular calcium concentration ([Ca²⁺]_i). Although Calu-3 cells lack functional purinergic receptors, a 33% hypotonic challenge triggered an [Ca²⁺]_i elevation in cells bathed bilaterally in nominally calcium free buffer (fura-2 fluorescence Δ340/380 = 0.509±0.114, n=10). BAPTA treatment of these cells abolished hypotonicity-induced [Ca²⁺]_i elevations (fura-2 fluorescence Δ340/380 = 0.026±0.016, n=10) and significantly reduced ATP release (223.1±20.3 nM and 607.8±43.1 nM in BAPTA and vehicle treated, respectively, n=6, P<0.05). Bafilomycin A₁ treatment significantly reduced hypotonicity-induced ATP release (341.7±33.6 nM and 615.8±55.3 nM in bafilomycin A₁ and vehicle treated, respectively, n=6, P<0.05). These observations suggest the involvement of a calcium-dependent pathway, possibly via exocytosis, in swelling-induced ATP release. However, elevation of [Ca²⁺]_i without cellular swelling, i.e. by thapsigargin or ionomycin, resulted in an increase in ATP release to 2- to 5-fold higher than basal levels (27.2±4.8, 21.7±4.1 and 6.9±0.6 nM in thapsigargin, ionomycin and vehicle treated, respectively, n=6). Thus, Ca²⁺_i partially regulated swelling-induced ATP release, but [Ca²⁺]_i elevation itself without cellular swelling was not sufficient to trigger maximal ATP release in Calu-3 cells.