Different truncated and conformationally constrained analogs of corticotropin-releasing factor (CRF) were synthesized on the basis of the amino acid sequences of human/rat CRF (h/rCRF), ovine CRF (oCRF), rat urocortin (rUcn), or sauvagine (Svg) and tested for their ability to displace [125I-Tyr0]oCRF or [125I-Tyr0]Svg from membrane homogenates of human embryonic kidney (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 (rCRFR1), or mouse CRF receptor, type 2beta (mCRFR2beta). Furthermore, the potency of CRF antagonists to inhibit oCRF- or Svg-stimulated cAMP production of transfected HEK 293 cells expressing either rCRFR1 (HEK-rCRFR1 cells) or mCRFR2beta (HEK-mCRFR2beta cells) was determined. In comparison with astressin, which exhibited a similar affinity to rCRFR1 (Kd = 5.7 +/- 1.6 nM) and mCRFR2beta (Kd = 4.0 +/- 2.3 nM), [DPhe11,His12]Svg(11-40), [DLeu11]Svg(11-40), [DPhe11]Svg(11-40), and Svg(11-40) bound, respectively, with a 110-, 80-, 68-, and 54-fold higher affinity to mCRFR2beta than to rCRFR1. The truncated analogs of rUcn displayed modest preference (2- to 7-fold) for binding to mCRFR2beta. In agreement with the results of these binding experiments, [DPhe11, His12]Svg(11-40), named antisauvagine-30, was the most potent and selective ligand to suppress agonist-induced adenylate cyclase activity in HEK cells expressing mCRFR2beta.