Endomorphin-1 (Tyr-Pro-Trp-Phe-NH 2) is a highly selective and potent agonist of the μ-opioid receptor. To identify structural attributes unique to this opioid peptide and potential sites of recognition, a conformational analysis has been performed using multidimensional NMR and molecular modeling techniques. The spectroscopic results, derived from experiments in both DMSO and water, indicate that endomorphin-1 exists in the cis- and trans-configuration with respect to the Pro-omega bond in approximately 25% and 75% populations, respectively. In DMSO, the cis-configuration adopts a compact sandwich conformation in which the Tyr and Trp aromatic rings pack against the proline ring, whereas the trans-configuration adopts an extended conformation. Although non-random structure was not observed in water, condensed phase molecular dynamics calculations indicate that trans-isomers dominate the population in this higher dielectric medium. Structural comparison of the cis- and trans-configurations with morphine and selective μ-peptide ligands PL-017 and d-TIPP, as well as the δ-selective peptide ligands TIPP (δ-antagonist, μ-agonist) and DPDPE were also performed and suggest the trans-isomer is likely the bioactive form. A hypothesis is proposed to explain μ- and δ-selectivity based on the presence of spatially distinct selectivity pockets among these ligands.