Astrocytes in the brain form an intimately associated network with neurons. They respond to neuronal activity and synaptically released glutamate by raising intracellular calcium concentration ([Ca2+]i), which could represent the start of back-signalling to neurons. Here we show that coactivation of the AMPA/kainate and metabotropic glutamate receptors (mGluRs) on astrocytes stimulates these cells to release glutamate through a Ca2+-dependent process mediated by prostaglandins. Pharmacological inhibition of prostaglandin synthesis prevents glutamate release, whereas application of prostaglandins (in particular PGE2) mimics and occludes the releasing action of GluR agonists. PGE2 promotes Ca2+-dependent glutamate release from cultured astrocytes and also from acute brain slices under conditions that suppress neuronal exocytotic release. When applied to the CA1 hippocampal region, PGE2 induces increases in [Ca2+]i both in astrocytes and in neurons. The [Ca2+]i increase in neurons is mediated by glutamate released from astrocytes, because it is abolished by GluR antagonists. Our results reveal a new pathway of regulated transmitter release from astrocytes and outline the existence of an integrated glutamatergic cross-talk between neurons and astrocytes in situ that may play critical roles in synaptic plasticity and in neurotoxicity.