Visualization of the release of an excitatory neurotransmitter, glutamate (Glu), from a slice preparation of the brain and spinal cord may be of great advantage in studying the release of Glu from a small population of neurons. When capsaicin (10 mu M) was applied to a slice of the rat spinal cord immersed in a medium containing glutamate dehydrogenase (GDH), an oxidized form of nicotinamide adenine dinucleotide (NAD+), and tetrodotoxin, we observed an apparent increase of fluorescence in superficial laminae and lamina X using a confocal laser scanning microscope. Such an increase was not observed in the absence of either NAD+ or GDH, was inhibited by removal of extracellular Ca2+, and was terminated by capsazepine (100 mu M). In contrast to capsaicin, Glu release evoked by high K+ was observed in all laminae throughout the grey matter. The present results suggest that this system enables us to see the site of the release of Glu as an image and that capsaicin releases this amino acid mainly in superficial laminae and lamina X in the spinal cord.