A novel cDNA clone encoding a putative G protein-coupled receptor (named GPCR21) was isolated from a mouse brain cDNA library along with its homologue, GPCR01 (the mouse counterpart of previously reported rat receptor R334 [(1991) FEBS Lett. 292, 243-248]) by the polymerase chain reaction using degenerate oligonucleotide primers. Northern blotting and reverse transcription-polymerase chain reaction analyses showed predominant expression of these two receptors in the central nervous system. In situ hybridization analysis revealed their prominent expression in the limbic system and further demonstrated the differential distribution of their mRNAs in mouse brain. Although the ligands for these receptors are yet to be identified, the significant sequence homology between these receptors suggests that they constitute a new receptor subfamily and they possibly represent different receptor subtypes for an unknown neurotransmitter.