Serotonin mediates various central and peripheral functions by its ability to bind to and activate a variety of receptors that belong to four pharmacologically defined classes termed 5-HT1 to 5-HT4. Using a polymerase chain reaction-derived probe, a clone, GPRFO, was isolated from a rat brain cDNA library. This cDNA encodes a putative G protein-coupled receptor that exhibits highest similarity to receptors for biogenic amines. RNA blot analysis indicated that the corresponding gene is expressed in the diencephalon. Consistent with the RNA blot data, GPRFO mRNA has been detected by in situ hybridization in the centrolateral, central medial, and intermediodorsal thalamic nuclei. Highest amounts of GPRFO mRNA, however, have been observed in a small area, i.e., the hippocampal rudiments and the stria longitudinalis. COS-7 cells transiently transfected with the GPRFO cDNA acquire saturable high-affinity binding sites for [3H]serotonin (KD = 41 nM). The pharmacological properties of the receptor differ from those of the known serotonin receptor subtypes, suggesting that the GPRFO cDNA encodes a novel serotonin receptor that is expressed in distinct rat brain regions.