ATP, a gliotransmitter released from astrocytes, works in glia-glia and glia-neuron communications. Still, the functional significance of astrocyte-derived ATP remains to be elucidated, since there has been no efficient and selective way to inhibit the ATP release. While two types of ATP release from astrocytes are known: vesicular and channel-mediated ATP release, the mechanisms of the channel-mediated ATP release are still unknown. To investigate those, we established a method that can directly detect single ATP release events from single astrocytes: ATP release was evoked by pressing cultured astrocytes with a glass pipette (mechanical stimulation), and released extracellular ATP was detected using GRAB-ATP_1.0, a protein ATP sensor together with the changes in intracellular Ca²⁺ with a Ca²⁺ indicator, Fura-2. Extracellular ATP and intracellular Ca²⁺ were observed to increase in synchrony. Through pharmacological screening with inhibitors, maxi-anion channels, connexin hemichannels, and volume-regulated anion channels revealed as candidates involved in astrocytic ATP release. Further analysis will provide detailed mechanisms and functions of ATP release from astrocytes.