We have utilized the polymerase chain reaction technique to selectively amplify a G protein-coupled receptor cDNA from rat kidney proximal convoluted tubule mRNA, which exhibits high homology with previously cloned serotonin receptors. Sequencing of a full-length clone isolated from a rat hippocampal cDNA library revealed an open reading frame of 1,212 base pairs encoding a 404-residue protein with seven hydrophobic regions predicted to represent transmembrane-spanning domains. Within the transmembrane regions, this receptor was found to be 44-50% identical with various members of the 5-HT1, 5-HT5, and 5-HT6 subfamilies with lower (37-40%) homology to the 5-HT2-like receptors. Northern blots revealed a approximately 3.6-kilobase transcript localized in various brain regions with the following rank order of abundance: hypothalamus > hippocampus = mesencephalon > cerebral cortex = olfactory bulb > olfactory tubercle. Expression of this clone in COS-7 cells resulted in the appearance of high affinity, saturable binding of [3H]lysergic acid diethylamide ([3H]LSD; KD = 5 nM) and [3H]serotonin ([3H]5-HT; KD = 1 nM). Among endogenous biogenic amines, only 5-HT completely inhibited radioligand binding. The inhibition of radioligand binding by other serotonergic agents revealed a pharmacological profile that does not correlate with any previously described serotonin receptor subtype. In addition, this receptor exhibits high affinity for a number of tricyclic antipsychotic and antidepressant drugs including clozapine, loxapine, and amitriptyline. In HEK-293 cells stably transfected with this receptor, serotonin elicits a potent stimulation of adenylylcyclase activity. The distinct structural and pharmacological properties of this receptor suggests that it represents a completely novel serotonin receptor subtype, which we propose to designate 5-HT7. Based on its pharmacology and its localization to limbic and cortical regions of the brain, it is likely that this receptor may play a role in several neuropsychiatric disorders that involve serotonergic systems.