Glutamate receptors (GluRs) mediate excitatory neurotransmission and may have important roles in central nervous system disorders. To characterize the human GLUR5 gene, which is located on human chromosome 21q22.1, we isolated cDNAs, genomic phage lambda clones, and yeast artificial chromosomes (YACs) and developed sequence tagged sites (STSs) and simple sequence length polymorphisms (SSLPs) for GLUR5. Genetic mapping with a tetranucleotide AGAT repeat named GLUR5/AGAT (six alleles observed, 70% heterozygosity) placed GLUR5 5 cM telomeric to APP (D21S210) and 3 cM centromeric to SOD1 (D21S223). The human GLUR5 gene is located near the familial amyotrophic lateral sclerosis (FALS) locus; linkage analysis of GLUR5 SSLPs in FALS pedigrees yielded negative lod scores, consistent with the recent association of the FALS locus with the SOD1 gene. Physical mapping of GLUR5 using a YAC contig suggested that the GLUR5 gene spans approximately 400-500kb, and is within 280kb of D21S213. The large size of the GLUR5 gene raises questions regarding its functional significance. Our GLUR5 YAC contig includes clones found in the Genethon chromosome 21 YAC contig, and reference to the larger contig indicates the orientation centromere--D21S213-GLUR5 5' end-GLUR5/AGAT--GLUR5 3' end--SOD1. The development of GLUR5/AGAT should permit rapid determination of the status of the GLUR5 gene in individuals with partial trisomy or monosomy of chromosome 21. Such studies may provide insights concerning the possible role of GLUR5 in Down syndrome.