The glycine receptor of the human CNS comprises ligand-binding alpha 1 and structural beta subunits encoded by the GLRA1 and GLRB genes, respectively. Screening of a human hippocampal cDNA library resulted in the identification of the novel subunit transcript beta B, differing in the 5'-UTR. Analysis of the genomic organization of GLRB showed that the coding region is distributed over nine exons, highly homologous to the GLRA1 gene. By in situ hybridization, the chromosomal localization of GLRB was refined to band 4q31.3. Based on the identical phenotypes of mouse lines carrying mutant alleles of the alpha 1 and beta subunit genes, GLRB was assumed to be a candidate gene for those cases of hyperekplexia that cannot be associated with mutations of GLRA1. Therefore, flanking intronic sequences were determined, and DNA samples from more than 30 index patients were subjected to SSCP screening of the entire GLRB coding region. A polymorphism in exon 8 was found both in the normal population and in families affected by hyperekplexia, although no coding mutation was detectable.