The existence of specific adrenomedullin receptor binding sites was investigated using the agonist peptide fragment [125I]human adrenomedullin-(13-52) in rat brain, lung and vas deferens homogenates. Saturation-binding experiments suggest that [125I]human adrenomedullin-(13-52) binds to an apparent single population of sites with similar affinities (K(D) of 0.3 to 0.6 nM) but with different maximal binding capacity in the rat brain, lung and vas deferens homogenates (B(max) of 73, 1760 and 144 fmol/mg protein, respectively). Competition-binding experiments using various analogues and fragments of calcitonin gene-related peptide (CGRP) and adrenomedullin were also performed using this radioligand. Competition-binding profiles suggest the possible existence of heterogeneous populations of adrenomedullin receptor binding sites. For example, in rat brain, human adrenomedullin-(1-52) and human adrenomedullin-(13-52) competed against specific [125I]human adrenomedullin-(13-52) sites with competition curves best fitted to a two-site model. Additionally, human calcitonin gene-related peptide alpha (hCGRPalpha), [Cys(Et)(2,7)]hCGRPalpha and [[R-(R,(R*,S*)]-N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)-,1-Piperidinecarboxamide] (BIBN4096BS) competed against specific [125I]human adrenomedullin-(13-52) binding with profiles that were also best fitted to a two-site model. Furthermore, binding assays performed in the presence of GTPgammaS (100 microM) revealed that this compound inhibited 20% of specific [125I]human adrenomedullin-(13-52) sites in rat brain homogenates and competition curves of human adrenomedullin-(1-52) and [Cys(Et)(2,7)]hCGRPalpha against specific [125I]human adrenomedullin-(13-52) sites remained best fitted to a two-site model. Moreover, the existence of specific [125I]human adrenomedullin-(13-52) binding sites that are resistant to human adrenomedullin-(22-52) and human CGRP-(8-37) is suggested in the rat brain and vas deferens. Taken together, these data provide evidence for the possible existence of heterogeneous populations of adrenomedullin binding sites in rat brain and peripheral tissues.