Astrocytes, a non-neuronal cell in the central nervous system, participate in the purinergic signaling. As lowered ATP release from astrocytes is related to depressive-like behavior, basal ATP transmission is thought to play important roles in physiological brain function. Since a single astrocyte interacts with up to 140,000 synapses in rodents, spatial property of ATP release is essential to understand purinergic signaling within brain architecture, which is not well understood. Using two-photon microscopy, we investigated spatiotemporal properties of ATP release in astrocytes with a novel genetically encoded extracellular ATP sensor, GRAB_ATP. In neuron-astrocyte coculture, TTX insensitive ATP release events were detected. In acute slices in which astrocytes expressed GRAB_ATP sparsely by means of in utero electroporation, we also observed spontaneous ATP release events, which were suppressed by astrocyte specific toxin, fluorocitrate, and insensitive to TTX and vesicular release blocker, bafilomycin A1. Typical ATP release spread over 50­–200 µm² with concentration roughly ranged 0.5–5 µM. Simultaneous monitoring with intracellular calcium revealed that ATP release and Ca²⁺ event rarely co-occurred. In conclusion, our findings indicate that astrocytes spontaneously release ATP in acute slices via mainly non-vesicular Ca²⁺ independent pathway, which possiblly activates purinergic receptors in nearby hundreds of synapses.