A growing body of evidence proposes that glial cells have the potential to play a role as modulators of neuronal activity and synaptic transmission by releasing the neurotransmitter glutamate (Araque et al., 1999). We explore the spatial nature of glutamate release from astrocytes with an enzyme-linked assay system and CCD imaging technology. In the presence of glutamate, L- glutamic dehydrogenase (GDH) reduces NAD+ to NADH, a product that fluoresces when excited with UV light. Theoretically, provided that GDH and NAD+ are present in the bathing saline, the release of glutamate from stimulated astrocytes can be optically detected by monitoring the accumulation of NADH. Indeed, stimuli that induce a wave of elevated calcium among astrocytes produced a corresponding spread of extracellular NADH fluorescence. Treatment of cultures either with thapsigargin, to deplete internal calcium stores, or with the membrane-permeant calcium chelator BAPTA AM significantly decreased the accumulation of NADH, demonstrating that this fluorometric assay effectively monitors calcium-dependent glutamate release. With a temporal resolution of 500 msec and spatial resolution of ~20 μm, discrete regions of glutamate release were not reliably resolved. The wave of glutamate release that underlies the NADH fluorescence propagated at an average speed of ~26 μm/sec, correlating with the rate of calcium wave progression (10-30 μm/sec), and caused a localized accumulation of glutamate in the range of 1- 100 μ. Further analysis of the fluorescence accumulation clearly demonstrated that glutamate is released in a regenerative manner, with subsequent cells that are involved in the calcium wave releasing additional glutamate.