[D-Pen(2), D-Pen(5)]enkephalin (DPDPE), a cyclic, constrained, highly potent, and delta opioid receptor-selective analogue of enkephalin, has been obtained from an aqueous solution in a crystalline form suitable for X-ray analysis. It crystallizes in the triclinic space group P1. The unit cell contains three conformationally distinct molecules of DPDPE which are located with approximate 3-fold symmetry about a water channel made up of approximately 24 disordered and one ordered water molecules. There are also 13 ordered water molecules which form an intricate network of hydrogen bonds which hold the peptide molecules together in the crystal. The conformation of the 14-membered ring is essentially identical for all three molecules; however, the Tyr-1 residue is conformationally different in each case. Comparison of the conformations found in the crystal with those previously determined by NMR methods in conjunction with energy calculations indicates that the most favorable conformation of the 14-membered ring in aqueous solution is similar to that in the crystal. This was interpreted to be due to the cyclic constraint in DPDPE and the high degree of solvation in the crystal structure. In addition, low-energy conformations previously determined by computational methods in attempts to determine the binding conformations of DPDPE gave conformations of the 14-membered rings which were generally similar to those found in the crystal structure. These results and previous structure-activity relationships suggest that the solid-state conformations are a useful starting point for understanding the bioactive conformation important for biological activity and delta receptor selectivity of cyclic enkephalin analogues.