The preferred conformations of the orphan G-protein coupled receptor agonists (des-bromo) neuropeptide B [1-23] and neuropeptide W [1-23], referred to as NPB and NPW, have been determined by ¹H NMR, CD, and molecular modeling. The sequences of NPB and NPW are WYKPAAGHSSYSVGRAAGLLSGL and WYKHVASPRYHTVGRAAGLLMGL, respectively. These are hypothalamic peptides that exert their biological actions on GPR7 and GPR8 receptors. Micellar solutions using the membrane mimetic, sodium dodecylsulphate-d₂₅ (SDS), were used to mimic a physiological environment for the peptides. The secondary structure of NPB consists of a type II β-turn involving residues Lys³ to Ala⁶. The C-terminal region of NPB exists in a conformational equilibrium between different secondary structures, including an α-helix from residues Arg¹⁵ to Ser²¹, and a 3₁₀-helix from residues Ser¹² to Ser²¹. The N-terminus of NPW exhibits a cation-π interaction between the Lys³ side chain and the quadrupole moment of the Trp¹ indole group. At the C-terminus of NPW, a well-defined α-helical conformation exists from Arg¹⁵ to Met²¹. As NPB and NPW have 91% sequence homology from residues Val¹³ to Leu²³, with only residue 21 differing between the two peptides, the similar C-terminal secondary structures of these two peptides are consistent with the sequences. This is supported by the similar CD spectra. The different secondary structures at the N-termini for NPB and NPW point to the importance of the N-terminus in receptor binding. This is consistent with the work of Fujii et al. [J. Biol. Chem. 277, 34010-34016 (2002)] who observed that iodination of the NPB Tyr² resulted in decreased agonistic activity at GPR7. In addition, Tanaka et al. [Proc. Natl. Acad. Sci. USA 100, 6251-6256 (2003)] showed that deletion of Trp¹ from NPB or NPW drastically decreased activity at GPR7 for NPB and GPR7 and GPR8 for NPW. Therefore, we postulate that the N-terminus is involved in membrane recognition and receptor binding.