The third extracellular loop of the human δ-opioid receptor (hDOR) is known to play an important role in the binding of 8-selective ligands. In particular, mutation of three amino acids (Trp284, Val296, and Va1297) to alanine significantly diminished δ-opioid receptor affinity for δ-selective ligands. To assess the changes in conformation accompanying binding of the endogenous opioid peptide deltorphin II to the δ-opioid receptor at both the receptor and ligand levels as well as to determine points of contact between the two, an in-depth spectroscopic study that addressed these points was initiated. Fragments of the δ-opioid receptor of variable length and containing residues in the third extracellular loop were synthesized and studied by NMR and CD spectroscopy in a membrane-mimetic milieu. The receptor peptides examined included hDOR-(279&ndsh;299), hDOR-(283– 299), hDOR-(281–297), and hDOR-(283–297). A helical conformation was observed for the longest receptor fragment between Val283 and Arg291, whereas a nascent helix occurred in a similar region for hDOR-(281–297). Further removal of N-terminal residues Val281 and Ile282 abolished helical conformation completely. Binding of the δ-selective ligand deltorphin II to hDOR-(279–299) destabilized the helix at the receptor peptide N terminus. Dramatic changes in the α-proton chemical shifts for Trp284 and Leu286 in hDOR-(279–299) also accompanied this loss of helical conformation. Large upfield displacement of α-proton chemical shifts was observed for Leu295, Va1297, and Va1297 in hDOR-(279–299) following its interaction with deltorphin II, thus identifying a gain in β-conformation at the receptor peptide C terminus. Similar changes did not occur for the shorter peptide hDOR(281–297). A hypothesis describing the conformational events accompanying selective deltorphin H binding to the δ-opioid receptor is presented. [ABSTRACT FROM AUTHOR]