Serotonin modulates a wide range of physiological functions by activating multiple receptors, which are coupled to various effector systems. Using a strategy based on amino acid sequence homology between 5-hydroxytryptamine (5-HT) receptors, we have isolated from a mouse brain library a cDNA encoding a new 5-HT receptor, 5-HTx, that activates adenylate cyclase. Amino acid sequence comparisons revealed that the 5-HTx receptor was a distant relative of previously cloned 5-HT receptors, with the highest percentage of homology (42%) being with the 5-HTdro1 receptor, a Drosophila 5-HT receptor positively coupled to adenylate cyclase. In COS-7 cells transiently expressing the 5-HTx receptor, 5-HT induced an increase in cAMP levels that was dose dependent and saturable (EC50 = 45 nM). Agonists displayed the following rank order of potencies: 5-carboxamidotryptamine > 5-methoxytryptamine > 5-HT > RU 24969 > 8-hydroxy-2-(di-n-propylamino)tetralin. The most efficient antagonists in inhibiting the stimulatory effect of 5-HT were methysergide, methiothepin, mesulergine, metergoline, clozapine, ergotamine, and (+)-butaclamol. Membranes of COS-7 cells expressing the 5-HTx receptor displayed a single saturable binding site for [3H]5-HT. The order of potencies of various drugs in displacing [3H]5-HT binding was similar to the order obtained in cAMP experiments. The pharmacological profile of this receptor does not correspond to the profile of any of the classic 5-HT receptor subtypes. Expression of 5-HTx mRNA was highest in brainstem and lower in forebrain, cerebellum, intestine, and heart. The 5-HTx receptor might therefore correspond to 5-HT1-like receptors that have been shown to induce relaxation in porcine vena cava and guinea pig ileum as well as tachycardia in cat heart. The high affinity of the 5-HTx receptor for neuroleptic agents such as (+)-butaclamol and clozapine suggests also that this receptor might play a role in certain neuropsychiatric disorders.