The D-isomer of aspartate is efficiently transported by high-affinity Na⁺/K⁺-dependent glutamatetransporters and is an effective ligand of N-methyl-D-aspartate (NMDA) receptors. To facilitate analysisof the regulation of these proteins in their native membranes, we synthesized a photolabile analogue ofD-aspartate, 4-methoxy-7-nitroindolinyl-D-aspartate (MNI-D-aspartate). This compound was photolyzedwith a quantum efficiency of 0.09 at pH 7.4. Photorelease of D-aspartate in acute hippocampal slicesthrough brief (1 ms) UV laser illumination of MNI-D-aspartate triggered rapidly activating currents inastrocytes that were inhibited by the glutamate transporter antagonist DL-threo-mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-benzyloxyaspartic acid(TBOA), indicating that they resulted from electrogenic uptake of D-aspartate. These transporter currentsexhibited a distinct tail component that was ~2% of the peak current, which may result from the releaseof K⁺ into the extracellular space during counter transport. MNI-D-aspartate was neither an agonist noran antagonist of glutamate transporters at concentrations up to 500 mages/entities/mgr.gif">M and was stable in aqueous solutionfor several days. Glutamate transporter currents were also elicited in Bergmann glial cells and Purkinjeneurons of the cerebellum in response to photolysis of MNI-D-aspartate, indicating that this compoundcan be used for monitoring the occupancy and regulation of glutamate transporters in different brainregions. Photorelease of D-aspartate did not activate mages/gifchars/alpha.gif" BORDER=0>-amino-3-hydroxy-5-methyl-4-isoxazolepropionicacid (AMPA)/kainate receptors or metabotropic glutamate receptors (mGluRs) in neurons, but resulted inthe selective, but transient, activation of NMDA receptors in hippocampal pyramidal neurons; MNI-D-aspartate was not an antagonist of NMDA receptors. These results indicate that MNI-D-aspartate alsomay be useful for studying the regulation of NMDA receptors at excitatory synapses.