In this study, the genomic organization of the human metabotropic glutamate receptor subtype 3 (mGluR3) gene has been determined. We have identified two transcription initiation sites and the polyadenylation signal by using 5'-rapid amplification of cDNA ends (RACE) and 3'-RACE, respectively. The exon/intron organization of the human mGluR3 gene revealed the presence of 6 exons separated by 5 introns. The size of introns varied from 10.4 to 120 kbp that contained consensus sequences for repetitive elements such as Alu and long interspersed elements. A putative promoter region flanking the 5' sequence of exon 1 was identified by computer-aided analysis. The putative promoter region was characterized by the presence of a CAAT and GC box, and the absence of a TATA box or CpG islands. Several putative binding sites for transcription factors were also identified. In addition, we have isolated, from a mouse genomic library, part of the mouse mGluR3 gene and found it to correspond to exon 2 in the human mGluR3 gene. The mouse mGluR3 gene was then mapped by fluorescent in situ hybridization analysis to chromosome 5qA2.