The preferred conformation of orexin-B, an orphan G-protein coupled receptor agonist (the human sequence is RSGPPGLQGRLQRLLQASGNHAAGILTM-NH₂) has been determined by ¹H and ¹³C 2D NMR spectroscopy and molecular modeling. Orexin-B has been implicated in sleep-wakefulness and feeding regulation. The membrane mimetic, sodium dodecylsulphate-d₂₅ (SDS), was used to mimic a physiological environment for the peptide. The secondary structure of orexin-B in SDS consists of two helical sections; helix I spans Leu⁷ to Ser¹⁸ and helix II spans Ala²² to Leu²⁶. Helices I and II are believed to be involved in membrane binding, as is supported by the results of the spin label studies with 5-doxylstearic acid. Lee et al. (Eur. J. Biochem. 266, 831-839 (1999)) determined the [Phe¹]-orexin-B conformation in water solution by NMR and showed that helix II extends from Ala²³ to Met²⁸. The C-terminal dipeptide, Thr²⁷-Met²⁸, is unstructured is SDS, whereas in water it forms the end of helix II. The lack of apparent structure for Thr²⁷-Met²⁸ in SDS allows the dipeptide to have conformational freedom to interact with the receptor. The conformation of orexin-B can now be used to explain the Ala substitution mutagenesis experiments and the D-amino acid substitution experiments (S. Asahi et al., Bioorg. Med. Chem. Lett. 13, 111-113, 2003). Asahi et al. have shown that Ala substitution from Gly²⁴ to Met²⁸ or D-amino acid substitution from Ala²³ to Met²⁸ causes a significant reduction in the potency of orexin-B for both OX₁R and OX₂R receptors. We postulate that helix II is involved in membrane recognition, and its binding to the membrane is essential for Thr²⁷-Met²⁸ to adopt the correct receptor-binding conformation.