Neurotransmitters released at synapses mediate Ca2+ signaling in astrocytes in CNS grey matter. Here, we show that ATP and glutamate evoke these Ca2+ signals in white matter astrocytes of the mouse optic nerve, a tract that contains neither neuronal cell bodies nor synapses. We further demonstrate that action potentials along white matter axons trigger the release of ATP and the intercellular propagation of astroglial Ca2+ signals. These mechanisms were studied in astrocytes in intact optic nerves isolated from transgenic mice expressing enhanced green fluorescent protein (EGFP) under control of the human glial fibrillary acidic protein promoter (GFAP) by Fura-2 ratiometric Ca2+ imaging. ATP evoked astroglial Ca2+ signals predominantly via metabotropic P2Y1 and ionotropic P2X7 purinoceptors. Glutamate acted on both AMPA- and NMDA-type receptors, as well as on group I mGlu receptors to induce an increase in astroglial [Ca2+]i. The direct Ca2+ signal evoked by glutamate was small, and the main action of glutamate was to trigger the release of the “gliotransmitter” ATP by a mechanism involving P2X7 receptors; propagation of the glutamate-mediated Ca2+ signal was significantly reduced in P2X7 knock-out mice. Furthermore, axonal action potentials and mechanical stimulation of astrocytes both induced the release of ATP, to propagate Ca2+ signals in astrocytes and neighboring EGFP-negative glia. Our data provide a model of multiphase axon–glial signaling in the optic nerve as follows: action potentials trigger axonal release of ATP, which evokes further release of ATP from astrocytes, and this acts by amplifying the initiating signal and by transmitting an intercellular Ca2+ wave to neighboring glia. © 2008 Wiley-Liss, Inc.