Ca²⁺ is considered a key element in multiple steps during regulated exocytosis. During the postfusion phase, an elevated cytoplasmic Ca²⁺ concentration ([Ca²⁺])_c leads to fusion pore dilation. In neurons and neuroendocrine cells, this results from activation of voltage-gated Ca²⁺ channels in the plasma membrane. However, these channels are activated in the prefusion stage, and little is known about Ca²⁺ entry mechanisms during the postfusion stage. This may be particularly important for slow and nonexcitable secretory cells. We recently described a “fusion-activated“ Ca²⁺ entry (FACE) mechanism in alveolar type II (ATII) epithelial cells. FACE follows initial fusion pore opening with a delay of 200–500 ms. The site, molecular mechanisms, and functions of this mechanism remain unknown, however. Here we show that vesicle-associated Ca²⁺ channels mediate FACE. Using RT-PCR, Western blot analysis, and immunofluorescence, we demonstrate that P2X₄ receptors are expressed on exocytotic vesicles known as lamellar bodies (LBs). Electrophysiological, pharmacological, and genetic data confirm that FACE is mediated via these vesicular P2X₄ receptors. Furthermore, analysis of fluorophore diffusion into and out of individual vesicles after exocytotic fusion provides evidence that FACE regulates postfusion events of LB exocytosis via P2X₄. Fusion pore dilation was clearly correlated with the amplitude of FACE, and content release from fused LBs was accelerated in fusions followed by FACE. Based on these findings, we propose a model for regulation of the exocytotic postfusion phase in nonexcitable cells in which Ca²⁺ influx via vesicular Ca²⁺ channels regulates fusion pore expansion and vesicle content release.