ORL1 is an endogenous G protein-coupled receptor for neuropeptide nociceptin. [(R/K)(14), (R/K)(15)]nociceptin is a superagonist that strongly activates the ORL1 receptor. We have previously found that substituting with Trp can reproduce the potentiation induced by Arg or Lys at position 14. In the present study, in order to ensure the effect of Trp-substitution on the activities of [(R/K)(14), (R/K)(15)]nociceptin, we synthesized [W(14), (R/K)(15)]nociceptin and [(R/K)(14), W(15)]nociceptin. [W(14), (R/K)(15)]nociceptin was found to exhibit threefold higher binding activity and 10-fold greater potency in a functional [(35)S]GTPgammaS functional assay as compared to wild-type nociceptin. However, when only Trp was placed in position 15, the resulting analogues, [(R/K)(14), W(15)]nociceptin, showed only a moderate enhancement of binding and biological activity (2-3 fold in both). These results indicate that the placement of Trp at position 14, unlike at position 15, enhances in a synergistic fashion the interaction of nociceptin with the ORL1 receptor. The results indicate that specific interactions feasible for Arg/Lys and Trp in common must be there for aromatic residues in ORL1, thus forming a cation/pi interaction or pi/pi hydrophobic interaction. The necessity for a favorable electrostatic interaction appears strict in position 15.