Sequential polymerase chain reaction experiments were performed to amplify a unique sequence representing a guanine nucleotide-binding protein (G-protein)-coupled receptor from rat hypothalamic cDNA. Degenerate oligonucleotides corresponding to conserved amino acids from transmembrane domains III, V, and VI of known receptors [5-HT1A, 5-HT1C, and 5-HT2; 5-HT is serotonin (5-hydroxytryptamine)] were used as primers for the sequential reactions. The resulting product was subcloned and used to screen a rat genomic library to identify a full-length clone (MR77) containing an intronless open reading frame encoding a 366-amino acid seven-transmembrane domain protein. The human homolog was isolated, and its encoded protein had 93% overall amino acid identity with the rat sequence. Within the conserved transmembrane domains, the sequences exhibit approximately 52%, 59%, 65%, and 68% amino acid identity with the known rat 5-HT1A, rat 5-HT1B, rat 5-HT1D, and human 5-HT1E receptors, respectively. MR77 was subcloned into a eukaryotic expression vector system and expressed in CosM6 cells. Studies on broken cell preparations indicate that the expressed receptor exhibits 125I-labeled d-lysergic acid diethylamide (LSD) binding that can be displaced by serotonin but not by other biogenic amines. The specific binding is displaced by the selective 5-HT1D agonist sumatriptan but not by the mixed 5-HT1A/1D agonist 5-carboxyamidotryptamine. 125I-labeled LSD binding was competitively antagonized by the ergot alkaloids methysergide and ergotamine. HeLa cells transfected with the MR77 gene exhibited inhibition of adenylate cyclase in response to serotonin. MR77 is expressed at low levels throughout the brain, with the greatest expression in the cortex, hippocampus, and striatum. MR77 thus represents a 5-HT receptor of the 5-HT1 class, and we propose that, based on the pharmacological characterization, MR77 represents an additional 5-HT1E-like receptor.