Two rat calcitonin (CT) receptor isoforms; C1a and C1b, are identical except for the presence of a 37-amino acid insert in the second extracellular domain of C1b. The functional consequences of this insert were examined after stable expression of these receptors into HEK-293 cells. In binding competition studies, dissociation of [ 125 I]salmon CT ([ 125 I]sCT) from C1b cells was rapid and complete, in contrast to dissociation from C1a cells, which was slow and incomplete, as seen with other CT receptor preparations. In these studies, C1a receptors displayed high affinity for salmon CT (Kd, 0.5 ± 1.3 nM) and a slightly lower affinity for pig CT. Human CT competed more weakly for binding of [ 125 I]CT. Although the relative affinities of the ligands were maintained for C1b receptors, the affinity for sCT was lower (K d , 23 ± 2 nM) and pig CT was approximately 10-fold less potent than sCT. Human and rat CT failed to compete with [ 125 I]sCT even at 1 microM with the C1b receptor. Both receptors influence multiple effector systems, indicating coupling to multiple G-proteins. The CT peptides activated adenylate cyclase with relative efficacies consistent with the binding competition potencies. In addition, both receptor isoforms mediated a rapid increase in the levels of intracellular calcium after a CT challenge. These results show that an extracellular modification in the rat CT receptor results in altered ligand recognition as well as altered binding kinetics, but does not modify their ability to generate multiple second messengers.