Galanin, a 29-30-amino acid neuropeptide, is widely distributed in central and peripheral systems and mediates a variety of physiological functions. Pharmacological studies have suggested the existence of multiple receptor subtypes but only the type I (GalR1) galanin receptor has been cloned. Now we report the cloning by a combination of sib selection and rapid amplification of cDNA ends of a cDNA encoding a new galanin receptor (GalR2) from rat hypothalamus. The receptor is 372 amino acids in length and shares only 40% homology with the rat GalR1 receptor. It contains seven putative transmembrane domains with the amino and carboxyl termini being least identical to GalR1. Northern blot analyses revealed a 2-kilobase pair mRNA species distributed in several tissues, suggesting a broader functional spectrum than GalR1. 125I-Labeled human galanin binding to rat GalR2 receptor expressed in COS-1 cells was saturable (Kd = 0.59 nM) and could be displaced by galanin, several galanin fragments, and chimeric peptides. The pharmacological profiles of GalR1 and GalR2 receptors were distinguishable by galanin fragment(2-29), which bound the cloned GalR2 receptor with markedly higher affinity than the GalR1 receptor. Activation of the cloned receptor by galanin led to inhibition of forskolin-stimulated intracellular cAMP production. The cloning of this new receptor subtype should provide further insights into the mechanisms by which galanin mediates its diverse physiological functions. The identification of galanin(2-29) as a receptor-specific ligand should enhance the understanding of specificity of galanin-receptor interactions.