The stereochemical requirements for omega-opioid receptor binding of a series of linear peptide antagonists with a novel conformationally restricted Phe analogue (Tic) as a second residue were examined by using a variety of computational chemistry methods. The omega-opioid receptor analogues with significant affinity, Tyr-Tic-NH2(TI-NH2), Tyr-Tic-Phe-OH(TIP), Tyr-Tic-Phe-NH2(TIP-NH2), Tyr-Tic-Phe-Phe-OH(TIPP), Tyr-Tic-Phe-Phe-NH2)(TIPP-NH2), and the low affinity omega-opioid peptides Tyr-Pro-Phe-Pro-NH2(morphiceptin) and Tyr-Phe-Phe-Phe-NH2 (TPPP-NH2), were included in this study. The conformational profiles of these peptides were obtained by consecutive cycles of high and low temperature molecular dynamic stimulations, coupled to molecular mechanical energy minimization carried out until no new conformational minima were obtained. Comparing the results for TPPP-NH2 and TIPP-NH2, the presence of the conformationally restricted Tic residue did not greatly reduce the number of unique low energy conformations, but did allow low energy conformers involving cis bonds between the first two residues. The conformational libraries of these peptides were examined for their ability to satisfy the three key ligand components for receptor recognition already identified by previous studies of high affinity cyclic (Tyr1-D-Pen2-Gly3-Phe4-D-Pen5) enkephalin (DPDPE) type agonists: a protonated amine group, an aromatic ring, and a lipophilic moiety in a specific geometric arrangement. Two types of conformations common to the five high omega-opioid affinity L-Tic analogues were found that satisfied these requirements, one with a cis and the other with a trans peptide bond between the Tyr1 and Tic2 residues. Moreover, both the Tic2 and Phe3 residues could mimic the hydrophobic interactions with the receptor of the Phe4 moiety in the cyclic DPDPE type agonists, consistent with the appreciable affinity of both di- and tripeptides. The low omega-opioid receptor affinity of morphiceptin can be understood as the result of conformational preferences that prevent the fulfillment of this pharmacophore for recognition.