Release of neurotransmitters outside synapses has broad biological implications, particularly with regard communication between axons and glia. Previous research has shown that ATP and adenosine released from axons firing action potentials can regulate myelination by Schwann cells and oligodendrocytes. Regulation of myelination by impulse activity could contribute to nervous system development and plasticity, and nonvesicular, nonsynaptic release of ATP could also participate in signaling between axons and other glial cells, neurons, and vasculature. We have identified a mechanism for ATP release from axons through volume-activated anion channels (VAAC) that are activated by microscopic axon swelling during action potential firing. A combination of single-photon imaging of ATP release, together with imaging for intrinsic optical signals, intracellular Ca 21 , time-lapse video and confocal microscopy, is used to investigate action potential-induced nonsynaptic release of this neurotransmitter. ATP release from premyelinated DRG axons persists when bafilomycin or botulinum toxin are used to block vesicular release, whereas pharmacological inhibition of VAACs or preventing action potential-induced axon swelling inhibited ATP release and blocked activity-dependent signaling between axons and astrocytes. This nonvesicular, nonsynaptic communication could mediate diverse activity-dependent interactions between axons and nervous system cells in normal conditions, development, and disease.