Mechanical perturbations, including hypotonic cell swelling, evoke rapid ATP release from a variety of cell types. The mechanism(s) of such release from epithelial cells and the regulatory pathways remain obscure. Two prevailing models propose conductive release, via ATP-permeable channels, or exocytosis of ATP-filled vesicles (Lazarowski et al. 2003). In a previous study, we showed that hypotonicity-evoked ATP release from human lung epithelial A549, airway epithelial 16HBE14o^– cells and 3T3 fibroblasts does not involve gadolinium-sensitive channels or volume-sensitive, 5-nitro-2-(3-phenylpropylamino) benzoic acid-inhibitable anion channels. It was, however, tightly dependent on intracellular Ca²⁺ elevation and abolished in BAPTA-loaded cells, implicating vesicular exocytosis (Boudreault & Grygorczyk 2002, Boudreault & Grygorczyk 2004). In the present study, we explored, in more detail, modulation of ATP secretion via the intracellular Ca²⁺ and protein kinase C (PKC) signaling pathways in A549 cells. ATP release from confluent A549 cell monolayers was investigated using a custom-made flow-through chamber (Boudreault & Grygorczyk 2004). ATP content in the samples was measured by lucifersase-luciferin assay, and ATP calibration curves were corrected for any inhibitory effect of the tested drugs on luciferase reaction. Under control conditions, 50% hypotonic shock triggered transient ATP release that peaked at around 1 min 45 s, and declined to baseline during the next 15 min. The peak rate of ATP secretion was 401 ± 49 pmol min^-1 (10⁶ cells)^-1, while cumulative ATP release during 15 min was 1,142 ± 151 pmol (10⁶ cells)^-1 (n = 5). Removal of extracellular Ca²⁺ had no effect on ATP secretion, indicating that it depends entirely on Ca²⁺ release from intracellular stores. Thapsigargin (1 μM, 1 h), a sarco-endoplasmic reticulum Ca²⁺-ATPase inhibitor, caused 57% ± 15% reduction of the peak rate of ATP release (n = 4, 23% ± 3% decrease of total ATP released). Caffeine (10 mM), which depletes intracellular Ca²⁺ stores by activating ryanodine-sensitive Ca²⁺ release channels, diminished ATP release by 34% ± 9% (n = 4). Heparin (1 mg/ml, 30 min), known to interfere with the IP3 receptor channel, reduced the peak of ATP release by 18% ± 6% (n = 4). Precise evaluation of the heparin effect was, however, hampered by its strong interference with the luciferase reaction. The kinetics of ATP release were strongly affected by treatment with PMA (1 μM, 15 min), a PKC activator. The peak rate was reduced by 71% ± 4% (n = 4), but release did not decline to baseline as rapidly as in control, untreated cells, and remained significantly elevated for more than 10-15 min. As a result, total ATP released during the 15-min period was slightly enhanced to 128% ± 19% compared to control cells. Brefeldin A (10 μM, 2.5 h), which blocks protein secretion by disrupting the Golgi complex and vesicle trafficking, reduced the peak rate of ATP release by 53% ± 24% (n = 3), suggesting that protein transport vesicles contribute to ATP release. Together with previously-found complete inhibition of ATP release by low temperature (Grygorczyk & Boudreault 2004), our data strongly support the concept that Ca²⁺-dependent vesicular exocytosis is a major mechanism of cell swelling-induced ATP secretion from A549 epithelial cells. This secretion is modulated by PKC and Ca²⁺ release from intracellular stores, involving at least two types of Ca²⁺ release channels. It does not, however, require direct extrcellular Ca²⁺ entry.