In order to investigate the dynamics of glutamate as a neurotransmitter and to avoid a complication by its metabolism, we studied the uptake and release of labeled non-metabolizable D-isomers of aspartate and glutamate in cerebral cortical slices and synaptosome preparation from guinea-pigs. The rate of uptake of D-aspartate and glutamate was mutually inhibited in a non-competitive fashion, indicating that their uptake mechanisms are not exactly the same. By ouabain (0.05 mM), the uptake of D-aspartate and glutamate into synaptosome preparation was less inhibited than that into cerebral slices. In synaptosome preparation most of the preloaded D-aspartate and glutamate was released by high-potassium (50 mM) stimulation, whereas in cerebral slices only a slight release was observed. However, when the slices were superfused with a medium free of sodium ions, which are absolutely necessary for the uptake, after preloaded with the labeled amino acids in the standard medium, a distinct release of radioactivity was induced by high-potassium stimulation. This potassium-induced release corresponded to only about 20% of the radioactivity accumulated in the slices. The accumulation of D-aspartate and glutamate into cerebral slices was much larger on the basis of their protein content than that into synaptosome preparation, when a high concentration (1 mM) of the amino acids was added to the medium. These observations suggest that the uptake system of D-aspartate and glutamate in cerebral slices is quite different from that in synaptosome preparation, and that the accumulation into cerebral slices is mainly localized in glial cells. In vivo the glial cell uptake is probably more important in removing the released neurotransmitter glutamate.