Previous corticotropin releasing factor 1 (CRF1) receptor characterization has been performed using radiolabeled agonists, which bind predominantly the receptor-G-protein complex. The pharmacological profile of other receptor states, and their abundance, remain poorly characterized. Here we investigated the affinity states of the CRF1 receptor heterologously expressed in Ltk- cells and endogenously expressed in rat cerebellum. In L-CRF1 cell membranes, three agonist affinity states were detected: a very-high affinity receptor-G-protein complex state (eliminated by GTPgammaS) bound by [125I]sauvagine (43 pM, RG); a high affinity state insensitive to GTPgammaS bound by [125I]sauvagine (1.4 nM, termed RO); and a low affinity G-protein-uncoupled state detected by sauvagine displacement of [125I]astressin, a labeled antagonist (120 nM, R). The relative abundance of RG:RO:R was 18%:16%:66%. All three states were demonstrated in rat cerebellum with similar relative abundance (15%:16%:69%). The R state bound CRF with low affinity (270-330 nM), displayed a novel rank order of ligand affinity, and represented the majority of the receptor population in both receptor preparations. This study provides a framework to identify CRF1 receptor conformational states in various receptor preparations.