Imaging the spatial and time-resolved distribution of biologically active substances in cells and tissues has attracted much attention in view of obtaining direct information about intracellular and intercellular processes of biological signaling cascades. 1 Various imaging methods have been reported, including fluorescence spectroscopy, 2 atomic-force microscopy 3 and electron microscopy. 4 Many studies have been described on the imaging of dynamic changes in the intracellular concentration and spatial distribution of target molecules and ions which play key roles in signal cascades, such as Ca 2+ , 5-7 adenosine 3′,5′-cyclic monophosphate (cAMP), 8,9 NO 10 and protein kinase C. 11 The spatial distribution of membrane proteins, such as the N-methyl-D-aspartate (NMDA) receptor and its subunits in brain slices, has also been imaged by autoradiography 12 and in situ hybridization. 13-15 Extracellular neurotransmitters having fluorescent groups, such as serotonin and catecholamines, have been imaged by native fluorescence microscopy. 16-19 However, no studies have been reported on imaging of L-glutamate, an important neurotransmitter in the brain, because of a lack of highly sensitive and selective probes for the neurotransmitter. Recently, an enzymatic, fluorometric imaging of L-glutamate releases from retinal slices has been proposed based on the fluorescence of NADH converted from NAD by an enzyme reaction between L-glutamate and glutamate dehydrogenase. 20 In the present paper, we describe an enzyme-based method for color imaging of L-glutamate released in mouse-brain slices. Cells and tissues are optically transparent in visible regions and imaging with visible radiation provides a simple method for detecting signals from target substances. The principle of the present method is shown in Fig. 1. L-Glutamate oxidase (GluOx) 21-23 and horseradish peroxidase (HRP) 22,23 are immobilized on a poly-L-lysine coated cover slip by the bovine serum albumin (BSA)-glutaraldehyde method. Brain slices are immersed in a solution of the substrate, in the present case DA-64, and transferred onto the membranes. The enzymes spontaneously diffuse from the membrane into the extracellular solution, where L-glutamate triggers an enzyme reaction to convert DA-64 to Bindschedler's Green (abbreviated hereafter as BG). The amount of BG formed is in a stoichiometric relation with that of L-glutamate and, hence, the intensity of the green signal is proportional to the concentration of L-glutamate. In this approach, the membrane is a reservoir of enzymes, from which enzymes are continuously supplied, directed toward an extracellular solution of the brain slice. The first examples of color images for the spatial distribution of L-glutamate in 25