Transport systems specific for L-glutamate and L-aspartate play an important role in the termination of neurotransmitter signals at excitatory synapses. We describe here the structure and function of a 66-kDa glycoprotein that was purified from rat brain and identified as an L-glutamate/L-aspartate transporter (GLAST). A GLAST-specific cDNA clone was isolated from a rat brain cDNA library. The cDNA insert encodes a polypeptide with 543 amino acid residues (59,697 Da). The amino acid sequence of GLAST suggests a distinctive structure and membrane topology, with some conserved motifs also present in prokaryotic glutamate transporters. The transporter function has been verified by amino acid uptake studies in the Xenopus laevis oocyte system. GLAST is specific for L-glutamate and L-aspartate, shows strict dependence on Na+ ions, and is inhibited by DL-threo-3-hydroxy-aspartate. In situ hybridization reveals a strikingly high density of GLAST mRNA in the Purkinje cell layer of cerebellum, presumably in the Bergmann glia cells, and a less dense distribution throughout the cerebrum. These data suggest that GLAST may be involved in the regulation of neurotransmitter concentration in central nervous system.