Through design, synthesis, and biological testing of constrained gonadotropin releasing hormone (GnRH) antagonists, we are studying the structural requirements for biological activity. Here we describe the conformational analysis in solution of a highly potent, dicyclic GnRH antagonist, dicyclo(4-10/5,5’-8)[Ac-D-2Nal(1),D-pClPhe(2),D-3Pal(3),Asp(4),Glu(5)(Gly),D-Arg(6),Dbu(8),Dpr(10)]GnRH (i), using NMR spectroscopy. The dicyclic part of this molecule adopts a preferred conformation containing a type II beta turn around residues 5-6, nested with a type I’ beta turn around residues 6-7, and a type II beta-turn-like structure involving residue 9 and the side chain of residue 10, which is stabilized by hydrogen bonds between Leu(7) NH/Asp(4) CO, Dbu8 NHdelta/Glu(5) CO, and Dpr(10) NHgamma/Dbu(8) CO. This is a novel conformation that had not been observed previously in any constrained GnRH antagonist and is remarkably different from that found for another dicyclic (4-10/5-8) GnRH antagonist with very similar sequence, dicyclo(4-10/5-8)[Ac-D-2Nal(1),D-pClPhe(2),D-Trp(3),Asp(4),Glu(5),D-Arg(6),Lys(8),Dpr(10)]GnRH (2) (Bienstock et al. J. Med. Chem. 1993, 36, 3265-3273). The conformation of 2 contains a type II’ beta turn around residues 6-7, which had been proposed to be essential for GnRH activity. These results are important for our general understanding of polypeptide conformation, since they show that the dicyclo(4-10/5-8) backbone can adopt more than one family of conformations despite its dicyclic nature, and from the point of view of the design of GnRH antagonists, since they suggest that the presence of a turn around residues 6-7, rather than the type of beta turn, may be necessary for biological activity.