Extracellular ATP and its metabolites are potent paracrine modulators of lung alveolar cell function, including surfactant secretion and fluid transport, but the sources and mechanism of intra-alveolar ATP release remain unclear. To determine the contribution of gas-exchanging alveolar type 1 (AT1) and surfactant-secreting type 2 (AT2) cells to stretch-induced ATP release, we used quantitative real-time luminescence ATP imaging and rat primary alveolar cells cultured on silicon substrate for 2–7 days. When cultured on solid support, primary AT2 cells progressively transdifferentiated into AT1-like cells with ~20% of cells showing AT1 phenotype by day 2–3 (AT2:AT1 ≈ 4:1), while on day 7, the AT2:AT1 cell ratio was reversed with up to 80% of the cells displaying characteristics of AT1 cells. Stretch (1 s, 5–35%) induced ATP release from AT2/AT1 cell cultures, and it was highest on days 2 and 3 but declined in older cultures. ATP release tightly correlated with the number of remaining AT2 cells in culture, consistent with ~10-fold lower ATP release by AT1 than AT2 cells. ATP release was unaffected by inhibitors of putative ATP channels carbenoxolone and probenecid but was significantly diminished in cells loaded with calcium chelator BAPTA. These pharmacological modulators had similar effects on stretch-induced intracellular Ca²⁺ responses measured by Fura2 fluorescence. The study revealed that AT2 cells are the primary source of stretch-induced ATP release in heterocellular AT2/AT1 cell cultures, suggesting similar contribution in intact alveoli. Our results support a role for calcium-regulated mechanism but not ATP-conducting channels in ATP release by alveolar epithelial cells.