Glial [Ca2+]i signaling was examined in a mammalian white matter lacking neuronal cell bodies and synapses. Rat optic nerves (postnatal days 2 and 7) were stained with calcium indicator dyes and confocal images of [Ca2+bdi were recorded at approximately 25 degrees C or approximately 37 degrees C. Glial cell bodies showed spiking or sustained [Ca2+], response to bath-applied glutamate (50–500 microM). The metabotropic glutamate agonist trans-ACPD elicited transient, sometimes spiking, [Ca2+], responses, whereas ionotropic agonists kainate and AMPA elicited a 6,7-dinitroquinoxaline-2,3-dione-sensitive, mostly sustained [Ca2+]i response. Transient and spiking glial [Ca2+]i responses also were elicited by adenosine and ATP (0.1–100 microM). Repetitive nerve stimulation (10–20 Hz) elicited [Ca2+bdi spiking in 15–25% of glial cells in postnatal day 7 nerves, with spiking typically occurring 15–60 sec after onset of nerve stimulation. At 37 degrees C, the frequency of glial [Ca2+]i spikes increased from approximately 0.06 Hz to approximately 0.11 Hz when axonal stimulation was increased from 10 to 20 Hz. This activity-dependent glial spiking was inhibited by TTX, could not be mimicked by increasing the bath K+ by 20 mM, and occurred when nerves were stimulated in the absence of bath calcium. Activity-dependent and glutamate-induced glial spiking could be mimicked by altering ionic gradients known to favor release of glutamate via glutamate transporters, including elevation of intracellular Na+ by veratridine concurrent with external K+ elevation. We suggest that glial [Ca2+]i spiking observed during electrical activity resulted from activation of glial receptors (e.g., metabotropic glutamate receptor, adenosine receptor) by substances (e.g., glutamate, adenosine) released from the optic nerve in a nonvesicular fashion, possibly through a reversal of sodium-coupled transporters when Na+ and K+ gradients are altered by prolonged nerve activity.