Antisauvagine-30 (aSVG) is the only high-affinity antagonist for the corticotropin-releasing factor (CRF) type 2 (CRF(2)) receptor. A structure-activity relationship study was performed to pinpoint residues conferring aSVG's selectivity. The aSVG-analogues being N-terminally extended by one or two residues or containing the Ala(22)Arg(23)Ala(24) (ARA-motif) of CRF, were synthesized. Additionally, a lactam bridge between positions 29 and 32 was introduced. The modified peptides were analyzed for alpha-helicity properties, binding affinities and antagonistic potencies at the rat CRF(1) and mouse CRF(2B) receptors. While N-terminal prolongation and replacement of D-Phe(11) by Tyr(11) increased the affinity for the CRF(2) receptor, the introduction of the ARA motif resulted in a loss of CRF(2) receptor selectivity. These data show that aSVG(10-40) analogues are more potent CRF(2) receptor antagonists than aSVG(11-40) peptides, while introduction of the ARA-motif or a cyclic constraint between residues 29 and 32 favors binding to the CRF(1) receptor.