Calcium signaling, manifested as intercellular waves of rising cytosolic calcium, is, in many cell types, the result of calcium-induced secretion of ATP and activation of purinergic receptors. The mechanism by which ATP is released has hitherto not been established. Here, we show by real-time bioluminescence imaging that ATP efflux is not uniform across a field of cells but is restricted to brief, abrupt point-source bursts. The ATP bursts emanate from single cells and manifest the transient opening of nonselective membrane channels, which admits fluorescent indicators of < or = 1.5 kDa. These observations challenge the existence of regenerative ATP release, because ATP efflux is finite and restricted to a point source. Transient efflux of cytosolic nucleotides from a subset of cells may represent a conserved pathway for coordinating local activity of electrically nonexcitable cells, because identical patterns of ATP release were identified in human astrocytes, endothelial cells, and bronchial epithelial cells.