The mouse gene encoding glial high-affinity, Na+-dependent glutamate transporter Slc1a3 (GluT-1/GLAST) was isolated, and its structural organization was characterized. The gene appeared to exist as a single copy in the mouse genome and comprised 10 exons spanning more than 56 kilobases. The transcription initiation sites were mapped to positions 503, which is the first transcriptional point (defined as +1), 128 (+376), and 64 (+440) basepairs upstream of the 3'-end of exon 1 by primer extension. The 5'-flanking region of the mouse GluT-1 gene had a typical CCAAT box and a GC box but lacked a TATA box. These features of the promoter region were characteristic of housekeeping genes. The fusion plasmids containing approximately 4 kb of the 5'-flanking region (-3830 to +450) and the firefly luciferase gene induced a significant luciferase activity when transfected into COS-1 cells. Distal deletion of the 5'-flanking region, leaving 619 bp (-169 to +450), resulted in a marked decrease in luciferase activity in COS-1 cells, suggesting that a CCAAT box, which was positioned at -200, is necessary for the expression of this gene. In situ hybridization localized this gene to mouse chromosome 15A2. These structural features will lead to a better understanding of the regulatory mechanism of the expression of the GluT-1 gene by ischemia and will also provide a basis for future evolutionary comparisons with other neurotransmitter transporters.