Screening of a genomic DNA library with a portion of the cDNA encoding the γ-aminobutyric acid (GABA) receptor subunit rho₁ identified two distinct clones. DNA sequencing revealed that one clone contained a single exon from the rho₁ gene (GABBR1) while the second clone encompassed an exon with 96% identity to the rho₁ gene. Screening of a human retina cDNA library with oligonucleotides specific for the exon in the second clone identified a 3-kb cDNA with an open reading frame of 1395 bp. The predicted amino acid sequence of this cDNA demonstrates 30 to 38% similarity to α, β, γ, and δ GABA receptor subunits and 74% similarity to the GABA rho₁ subunit suggesting that the newly isolated cDNA encodes a new member of the rho subunit family, tentatively named GABA rho₂. Polymerase chain reaction (PCR) amplification of rho₁ and rho₂ gene sequences from DNA of three somatic cell hybrid panels maps both genes to human chromosome 6, bands q14 to q21. Tight linkage was also demonstrated between restriction fragment length variants (RFLVs) from each rho gene and the Tsha locus on mouse chromosome 4, which is homologous to the CGA locus on human chromosome 6q12–q21. These two lines of evidence confirm that GABRR1 and newly identified GABRR2 map to the same region on human chromosome 6. This close physical association and high degree of sequence similarity raises the possibility that one rho gene arose from the other by duplication.