We and others have recently cloned the genes encoding the human 5-HT(1D) (5-HT(1Dalpha)) and 5-HT(1B) (5-HT(1Dbeta)) serotonin receptors. Because of the history of profound species differences in the pharmacology of these receptor subtypes, we also cloned the homologous genes for these two receptors in rat. The rat 5-HT(1D) receptor gene, like that of the rat 5-HT(1B) receptor, is intronless, encoding a 374-amino acid polypeptide 90% identical to its human homologue. The rat 5-HT(1D) and rat 5-HT(1B) receptors are 61% identical in their deduced amino acid sequences. The availability of both the rat 5-HT(1B) and 5-HT(1D) genes allowed direct comparison of the pharmacological properties of the two receptors expressed in transfected cells as assessed using 5-[(3)H]HT binding assays. Unlike the rat 5-HT(1B) receptor, which is pharmacologically dimorphic with respect to its human homologue, the rat 5-HT(1D) receptor has an almost identical profile compared to the human 5-HT(1D) receptor. (+/-)-Cyanopindolol and (-)-propranolol are more than 100-fold selective for the rat 5-HT(1B) receptor over the rat 5-HT(1D) receptor, while mianserin is more than 100-fold selective for the rat 5-HT(1D) receptor. The rat 5-HT(1D) receptor is expressed in cells of the dorsal raphe and thus may serve as an additional type of serotonin autoreceptor. This constitutes the first unambiguous characterization of a 5-HT(1D) receptor in rat and demonstrates its relationship to the 5-HT(1B) receptor in rat as well as to the 5-HT(1D) and 5-HT(1B) receptors in human.