Two rat calcitonin receptor isoforms have been identified by cDNA cloning from a hypothalamic library. The clones, C1a and C1b, specified proteins of 478 and 515 amino acids, respectively. The clones were identical, except that the C1b sequence encoded a 37-amino acid insert in the second extracellular domain, which conferred altered ligand recognition. Compared to the C1a receptor, expressed C1b receptors exhibited decreased affinity for porcine CT, relative to salmon CT, and negligible affinity for human CT. Clone C1b mRNA was predominately expressed in the brain, whereas mRNA for the C1a clone was present in both brain and peripheral tissues. Both receptors were able to couple functionally to adenylate cyclase. Thus, clone C1b represents a novel brain isoform of the CT receptor with different affinity for CT analogs resulting from an altered second extracellular domain.