We describe the binding of [125I]tyr(o)sauvagine to membranes of corticotropin-releasing hormone (CRH2) receptor expressing HEK293/EBNA (293ECRH2 alpha) cells. The binding of [125I]tyr(o)sauvagine to CRH2 receptors was temperature, time and tissue dependent. Equilibrium was reached in 2 hr at 23 degrees C. Saturation data best fit a two-site model with affinity constants of 44 pM and 4.1 nM for high and low affinity states, respectively. The high affinity [125I]tyr(o)sauvagine binding sites were eliminated with 200 microM Gpp (NH) p, indicating coupling to G proteins. The rank order potency of peptide analogs of CRH to inhibit [125I]tyr(o)sauvagine binding to CRH2 alpha receptors was: urotensin > sauvagine = urocortin > alpha-helical CRH9-41 > rh-CRH >> o-CRH. This was in contrast to the rank order potency of the peptides at inhibiting [125I]tyr(o)oCRH binding to CRH, receptors: urotensin > urocortin > r/h-CRH > o-CRH = sauvagine > alpha-helical CRH9-41. We show that two recently identified small molecule CRH antagonists with nanomolar potency at the CRH1 receptor, have little or no affinity for CRH2 alpha receptors as labeled by [125I]tyr(o)sauvagine. Two selective CRH1 receptor antagonists enabled us to examine comparative densities of CRH1 and CRH2 receptors in several brain areas. We also used [125I]tyr(o)sauvagine in combination with a specific CRH1 antagonist to examine the anatomic distribution of CRH2 receptors using receptor autoradiography. With a few notable exceptions the CRH2 receptors demonstrated autoradiographically in this study match the data obtained by in situ hybridization studies on the localization of CRH2 mRNA. The anatomic overlap of the autoradiographic and in situ hybridization data suggest that CRH2 receptors are postsynaptic. This study demonstrates the utility of using [125I]tyr(o)sauvagine to study cloned CRH2 receptors expressed in cell lines. In addition, [125I]tyr(o)sauvagine used in conjunction with saturating concentrations of a specific CRH1 receptor antagonist allows the study of CRH2 receptors in brain tissues using both in vitro homogenate binding and receptor autoradiography techniques.