The conformational features of the delta-selective, cyclic opioid peptide Tyr-cyclo[D-Cys-Phe-D-Pen]OH (JOM-13) were investigated using a combination of experimental (X-ray crystallography, H-1 NMR spectroscopy) and theoretical (molecular mechanics computations) techniques. Energy calculations with the CHARMm force field show the existence of a single energetically preferred backbone conformation for the cyclic tripeptide portion of the molecule. Several distinct low-energy conformations were calculated, however, for the disulfide bridge linking the D-Cys and D-Pen residues, for the single Tyr residue outside the cycle, and for the Tyr and Phe side chains. The two calculated lowest-energy conformers of the D-Cys, D-Pen disulfide bridge (A and B) differ in values of D-Cys chi1 and chi3 (S-S) torsion angles (the -60-degrees,90-degrees and 180-degrees,-90-degrees combinations). This is consistent with the observation of two H-1 NMR signal sets in aqueous solution (in the ratio 68:32) with distinctly different vicinal coupling constants for protons H-C(alpha)C(beta)-H that correspond to a D-Cys chi1 angle of about -60-degrees for the first set and approximately 180-degrees or +60-degrees for the second. X-ray crystallographic analysis of crystals grown from aqueous solution also revealed two independent conformers which are in excellent agreement with the computational and NMR data for the rigid, cyclic part of the molecule including the disulfide bridge. However, H-1 NMR data and computational results indicate that the flexible elements of JOM-13 (the exocyclic Tyr residue and the Tyr and Phe side chains) have no fixed structure in water solution. In the crystalline environment they adopt conformations that are stabilized mainly by intermolecular interactions and which do not correspond exactly to any local energy minimum identified in the molecular mechanics calculations.