Ca²⁺ is the key element in regulated exocytosis controlling multiple steps during the exocytic pre- and post-fusion stages. We have recently described a novel "fusion-activated" Ca²⁺-entry during the postfusion stage of lamellar body exocytosis in alveolar type II (ATII) cells. In the present study we have identified the P2X4 receptor as the underlying molecular entity for the localised Ca²⁺-entry. Using RT-PCR, western-blotting and immunofluorescence we demonstrate for the first time that P2X4 is expressed in ATII cells and is primarily localised on lamellar bodies. Pharmacological and genetic data confirm that the localised Ca²⁺-entry following lamellar body exocytosis is mediated via vesicular P2X4 receptors. Furthermore, previously developed imaging techniques with high spatial and temporal resolution provide evidence that "fusion-dependent" Ca²⁺-entry via P2X4 regulates post fusion events of lamellar body exocytosis. We measured diffusion rates of fluorescent dyes through the fusion pore as direct indicators for fusion pore diameters. Fusion pore opening (diameter) clearly correlated with the amplitude of the "fusion-dependent" Ca²⁺-entry for individual lamellar body fusions. Moreover, we find a marked acceleration of content release from fused lamellar bodies when fusion was followed by "fusion-dependent" Ca²⁺-entry. We conclude that P2X4 receptors on lamellar bodies mediate a localised Ca²⁺-entry following lamellar body fusion with the plasma membrane and that this localised Ca²⁺ signal controls fusion pore expansion and subsequent surfactant release.