Using a strategy based on amino acid sequence homology between 5-hydroxytryptamine (5-HT) receptors that interact with G proteins, we have isolated from a mouse brain library a cDNA encoding a new serotonin receptor, the 5HT1E beta receptor. Amino acid sequence comparisons revealed that its closest relatives were the recently characterized 5HT1E receptor (S31) and the 5HT1B and 5HT1D receptors. When expressed transiently in Cos-7 cells, the 5HT1E beta receptor displayed a high affinity for the nonspecific serotonergic radioligand 2-[125I]iodolysergic acid diethylamide (Kd = 980 pM). The pharmacological profile of the 5HT1E beta receptor resembled that of previously reported 5HT1E sites that have a low affinity for 5-carboxamidotryptamine and that have been found in human and rat brain. When stably expressed in NIH-3T3 cells, the 5HT1E beta receptor was negatively coupled to adenylate cyclase. In situ hybridization experiments revealed that the 5HT1E beta transcripts were detected only in the CA1, CA2, and CA3 layers of the hippocampus. Our results therefore demonstrate that the 5HT1E receptors constitute a heterogeneous family of receptors.