We report the molecular cloning of a fragment of human genomic DNA called S12, containing an open reading frame of 1170 nucleotides, which encodes a receptor for serotonin of 390 amino acids. The receptor function of the S12 protein was demonstrated by functional expression in mouse LS12 cells obtained by stable transfection of Ltk- cells, and LM5S12 cells, derived from LM5 cells (Ltk- cells previously transfected with the M5 muscarinic acetylcholine receptor). Adenylyl cyclase studies showed that the S12 receptor is able to mediate inhibition of adenylyl cyclase in response to serotonin in both types of cells. As studied in LM5S12 cells, the S12 receptor did not promote Ca2+ mobilization from internal stores, nor did it significantly modulate the sustained increase in [Ca2+]i elicited by stimulation of the phospholipase C stimulating M5 acetylcholine receptor. The pharmacologic profile of S12 as seen in adenylyl cyclase assays is as follows: (EC50 in nM): serotonin, full agonist (37 nM), 5-carboxamidotryptamine, full agonist (10 nM), sumatriptan, full agonist (50 nM), metergoline, partial agonist (10 nM), methysergide, partial agonist (40 nM), yohimbine, partial agonist (150 nM), metitepin, antagonist (KB = 0.7 to 1.2 nM). We propose that the human S12 serotonin receptor is a receptor of the 5-hydroxytryptamine1D subtype.