Deltorphin-I, Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2 and dermenkephalin, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, two highly related opioid peptides from frog skin, display very similar N-termini but strikingly different C-terminal tails. Nevertheless, both peptides are highly potent at, and exquisitely selective for the delta-opioid receptor. To identify common determinants concuring to the remarkably efficient targeting of deltorphin-I and dermenkephalin, combined use of quantitative two-dimensional nuclear magnetic resonance (53 dipolar interactions studied at four temperatures) and energy calculations using simulated annealing generated five groups of deltorphin-I conformers. These groups were pooled into two families whose overall conformation could be described either by a left-handed helix (Family I) or by a big loop (Family II), both stabilized by H-bonds. Proximity of D-Ala2-Phe3-Asp4 and Val5-Val6-Gly7 triads is an obvious structural similarity between almost all groups in both families of structures. Whereas differences between the two families originated mostly from a transition at psi Asp4 backbone dihedral angle, the backbone structures at segment 1-4 are similar and spatial arrangements of Tyr1 (t) and Phe3 (g-) are identical in one group of each family. Moreover, these two groups have a N-terminal tetrapeptide whose conformation most closely resembles that of a well-defined group of structures for dermenkephalin. Altogether, these results suggest that conformational attributes that are common to dermenkephalin and deltorphin-I, i.e., the backbone conformation of the N-terminal tetrapeptide and preferential orientations in the side-chain of Tyr1 (t) and Phe3 (g-) underlie their ability to bind with high selectivity to the delta-opioid receptor.