Astrocyte glutamate release can modulate synaptic activity and participate in brain intercellular signaling. P2X₇receptors form large ion channels when activated by ATP or other ligands. Here we show that P2X₇ receptors provide a route for excitatory amino acid release from astrocytes. Studies were performed using murine cortical astrocyte cultures. ATP produced an inward current in patch-clamped astrocytes with properties characteristic of P2X₇ receptor activation: the current was amplified in low divalent cation medium, blocked by pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), and more potently activated by 3′-O-(4-benzoyl)benzoyl ATP (BzATP) than by ATP itself. Measurement of current reversal potentials showed the relative BzATP-induced permeabilities to different substrates to be Na⁺, 1 > Cl⁻, 0.34 >N-methyl-d-glucamine, 0.27 >l-glutamate, 0.15 ≈ d-aspartate, 0.16. Astrocytes exposed to BzATP also became permeable to Lucifer yellow, indicating a large channel opening. Release of l-glutamate and d-aspartate through P2X₇ channels was confirmed using radiolabeled tracers. As with the inward current, release of glutamate and d-aspartate was induced by BzATP more potently than ATP, amplified in Ca²⁺/Mg²⁺-free medium, and blocked by PPADS or oxidized ATP. Efflux through P2X₇channels is a previously unrecognized route of ligand-stimulated, nonvesicular astrocyte glutamate release.