The gene encoding a human 5-hydroxytryptamine (5-HT)1 receptor subtype was isolated from a human placental genomic library by using oligonucleotide probes derived from transmembrane regions of the cloned human 5-HT1D beta receptor. The deduced amino acid sequence of the genomic clone hp75d is identical to that of the recently isolated, but uncharacterized, novel serotonin receptor gene S31. Transmembrane domain sequence comparison of clone hp75d with other guanine nucleotide-binding protein-coupled receptors revealed the highest degree of homology (64%) to the 5-HT1D alpha and 5-HT1D beta subtypes and lower degrees of homology (35-52%) to other serotonergic and catecholaminergic receptors. A stable cell line expressing this gene was established, using murine fibroblasts as the host cell, for pharmacological evaluation. High affinity (Kd = 9.7 nM), saturable (Bmax = 2.4 pmol/mg of protein) [3H]5-HT binding was detected using membranes derived from stable transfectants. Most compounds displayed low affinity (K(i) greater than 200 nM) for the expressed gene, with the exception of 5-HT (K(i) = 10 nM). The rank order of potency of ligands to compete for the [3H]5-HT-labeled site best matched the binding profile of the pharmacologically defined 5-HT1E binding site, 5-HT greater than methysergide greater than ergotamine greater than 8-hydroxy-2-(di-n-propylamino)tetralin greater than 5-carboxyamidotryptamine greater than ketanserin. 5'-Guanylylimidodiphosphate decreased high affinity agonist ([3H]5-HT) binding in a dose-dependent manner. 5-HT produced a dose-dependent inhibition of forskolin-stimulated cAMP accumulation in intact cells stably expressing the 5-HT1E gene. The response was blocked by the nonselective 5-HT1 receptor antagonist methiothepin. The molecular biological and pharmacological data are consistent with the designation that clone hp75d encodes a functional 5-HT1E receptor.