Activation of purinoceptors by extracellular ATP is an important component of the glial response to injury in the central nervous system (CNS). ATP has been shown to evoke raised cytosolic [Ca2+] in astrocytes, oligodendrocytes, and microglia, the three major glial cell types in the CNS. Glial cells express a heterogenous collection of metabotropic P2Y and ionotropic P2X purinoceptors, which respectively mobilise Ca2+ from intracellular stores and trigger Ca2+ influx across the plasmalemma. It is likely that different receptors have distinct roles in glial cell physiology and pathology. Our studies on optic nerve glia in situ indicate that P2Y1 and P2Y2/4 receptors are activated at low ATP concentrations, suggesting they are the predominant purinoceptors mediating physiological Ca2+ signalling. Glia also express P2X1 and P2X3 purinoceptors, which mediate fast, rapidly desensitising current and may also be important in signalling. At high concentrations, such as occur in CNS injury, ATP induces large and prolonged increases in glial [Ca2+]i with a primary role for P2Y purinoceptors and inositol trisphosphate (IP3)-dependent release of Ca2+ from intracellular stores. In addition, we found that high concentrations of ATP activated a significant P2X component that did not desensitise or saturate and was dependent on extracellular Ca2+. These are characteristic properties of the P2X7 subtype, and we provide in situ evidence that application of the P2X7 receptor agonist benzoyl-benzoyl ATP (BzATP) evokes raised [Ca2+]i in optic nerve glia, and that the dye YO-PRO-1, which passes through pore-forming P2X7 receptors, is taken up by astrocytes, oligodendrocytes and microglia. Glia also express P2X2 and P2X4 receptors that are also pore-forming in the presence of sustained high ATP concentrations and which may also be important in the glial injury response. There is evidence that activation of P2 purinoceptors is a key step in triggering reactive changes in glial cells, including expression of immediate early genes, induction of extracellular signal regulated kinase and cyclooxygenase-2, synthesis of phospholipase A2, release of arachidonic acid, production of prostaglandins and release of interleukins. We show that the ATP-mediated increase in glial [Ca2+]i is potentiated by arachidonic acid and reduced by the inhibition of phospholipase A2 inhibition. Together, the results implicate ATP as a primary signalling molecule in glial cells and indicate specific roles for P2Y and P2X purinoceptors in glial cell pathology.