Serotonin [5-hydroxytryptamine (5-HT)] is a neuromodulator that mediates a wide range of physiological functions by activating multiple receptors. Using a strategy based on amino acid sequence homology between 5-HT receptors that interact with guanine nucleotide-binding proteins, we have isolated from a mouse brain library a cDNA encoding a new serotonin receptor. Amino acid sequence comparisons revealed that this receptor was a close relative of the previously identified 5-HT5 receptor but was distant from all other 5-HT receptor subtypes; we therefore named it 5-HT5B. When expressed in COS-7 cells, the 5-HT5B receptor displayed a high affinity for the serotonergic radioligand 125I-lysergic acid diethylamide. Its pharmacological profile was distinct from that of all classic 5-HT receptor subtypes. However, the high affinity of the 5-HT5B receptor for 5-carboxamidotryptamine and its low affinity for sumatriptan indicated that it might correspond to recently described 5-HT1D-like binding sites that were labeled with [3H]5-carboxamidotryptamine and insensitive to sumatriptan. In situ hybridization experiments revealed that the 5-HT5B mRNA was expressed predominantly in the habenula and in the CA1 field of the hippocampus. We also determined the chromosomal localization of the 5-HT5A and 5-HT5B genes and of their human counterparts. The 5-HT5A gene colocalized with the mouse mutation reeler and the human mutation holoprosencephaly type 3, which both result in abnormal brain development, raising the possibility that the 5-HT5A receptor plays a role in brain development.