Astrocytes release a variety of transmitter molecules, which mediate communication between glial cells in the brain and modulate synaptic transmission. ATP is a major glia-derived transmitter, but the mechanisms and kinetics of ATP release from astrocytes remain largely unknown. Here, we combined epifluorescence and total internal reflection fluorescence microscopy to monitor individual quinacrine-loaded ATP-containing vesicles undergoing exocytosis in cultured astrocytes. In resting cells, vesicles exhibited three-dimensional motility, spontaneous docking and release at low rate. Extracellular ATP application induced a Ca(2+)-dependent increase in the rate of exocytosis, which persisted for several minutes. Using UV flash photolysis of caged Ca(2+), the threshold Ca(2+) for ATP exocytosis was found to be approximately 350 nM. Subthreshold Ca(2+) transients predominantly induced vesicle docking at plasma membrane without subsequent release. ATP exocytosis triggered either by purinergic stimulation or by Ca(2+) uncaging occurred after a substantial delay ranging from tens to hundreds of seconds, with only approximately 4% of release occurring during the first 30 s. The time course of the cargo release from vesicles had two peaks centered on <or=10 s and 60 s. These results demonstrate that: (1) Ca(2+) elevations in cultured astrocytes trigger docking and release of ATP-containing vesicles; (2) vesicle docking and release have different Ca(2+) thresholds; (3) ATP exocytosis is delayed by several minutes and highly asynchronous; (4) two populations of ATP-containing vesicles with distinct (fast and slow) time course of cargo release exist in cultured astrocytes.