Adrenomedullin (ADM) is a hypotensive peptide with structural homology, including a ring structure linked by a disulfide bridge, to calcitonin gene-related peptide (CGRP), calcitonin and amylin. ADM is predominantly synthesized in the adrenal medulla, but immunoreactive ADM has also been detected in the human brain. Here we have characterized ADM binding sites in cultured rat astrocytes using human [125I]ADM(1-52) as radioligand. Half-maximal inhibition of [125I]ADM(1-52) binding by intact rat ADM(1-50) amounted to 0.27 +/- 0.03 nM (n = 15). The related peptides rat alpha-CGRP, rat amylin and salmon calcitonin displaced [125I]ADM(1-52) at 85-, 148-, and > 4000-fold higher concentrations. Half-maximal stimulation of cAMP accumulation by rat ADM(1-50) was obtained with 1.00 +/- 0.12 nM (n = 16). Rat alpha-CGRP was 214-fold, and rat amylin and salmon calcitonin were > 1000-fold less potent. Concerning cAMP accumulation the results were indistinguishable in mouse neuroblastoma x rat glioma hybrid cells (NG108-15), but here rat alpha-CGRP was > 1000-fold less potent than rat ADM(1-50). Human ADM(22-52) and human CGRP-I(8-37), which lack the ring structure, failed to stimulate cAMP accumulation, but they antagonized rat ADM(1-50) stimulated cAMP accumulation with inhibitory constants of 365 +/- 93 nM and 92 +/- 2 nM In astrocytes, and 45 +/- 3 nM and 1300 +/- 500 nM in NG108-15 cells. Rat ADM(1-50) did not raise cytosolic free calcium concentrations in astrocytes and NG108-15 cells. In conclusion, we have identified novel ADM receptors coupled to cAMP formation in cultured rat astrocytes and NG108-15 cells. Different interactions with the homologous peptide CGRP as well as truncated receptor antagonists ADM(22-52) and CGRP(8-37) in rat astrocytes and neuroblastoma x glioma hybrid cells are consistent with ADM receptor isotypes in the brain.