Modulation of synaptic transmission and brain microcirculation are new roles ascribed to astrocytes in CNS function. A mechanism by which astrocytes modify neuronal activity in the healthy brain depends on fluctuations of cytosolic Ca²⁺ levels, which regulate the release of “gliotransmitters” via an exocytic pathway. Under pathological conditions, however, the participation of other pathways, including connexin hemichannels and the pore-forming P2X₇R, have been proposed but remain controversial. Through the use of genetically modified 1321N1 human astrocytoma cells and of spinal cord astrocytes derived from neonatal Cx43- and P2X₇R-null mice, we provide strong evidence that P2X₇Rs, but not Cx43 hemichannels, are sites of ATP release that promote the amplification of Ca²⁺ signal transmission within the astrocytic network after exposure to low divalent cation solution. Moreover, our results showing that gap junction channel blockers (heptanol, octanol, carbenoxolone, flufenamic acid, and mefloquine) are antagonists of the P2X₇R indicate the inadequacy of using these compounds as evidence for the participation of connexin hemichannels as sites of gliotransmitter release.