c-[D-Pen(2),D-Pen(5)]enkephalin (DPDPE, 1) is a cyclic, constrained, highly potent, delta opioid receptor selective peptide agonist. Substitution of Gly(3) with L-Ala in DPDPE to give [L-Ala(3)]DPDPE (2) has been shown to produce a peptide with much greater delta receptor binding selectivity than DPDPE itself. However [L-Ala(3)]DPDPE is only a partial agonist in in vivo antinociception and actually was found to potently antagonize the antinociceptive effects of DPDPE at delta receptors in the brain. In comparison, [D-Ala(3)]DPDPE (3) is a weak and poorly selective delta agonist. In an effort to correlate the biological profiles of these peptides with secondary structure, [L-Ala(3)]DPDPE and [D-Ala(3)]DPDPE were studied by X-ray crystallography and H-1 and C-13 NMR in DMSO solution. Crystals of both peptides were obtained using vapor diffusion techniques. [L-Ala(3)]DPDPE crystallizes in the monoclinic space group C2 with cell dimensions a = 36.35(1) Angstrom, b = 19.737(4) Angstrom, c = 28.16(1) Angstrom, beta = 129.07(2)degrees, and V = 15688(9) Angstrom(3). The asymmetric unit contains four peptide molecules and approximately 20 water molecules, giving a calculated density of 1.274 g cm(-3). The conformation of all four independent [L-Ala(3)]DPDPE molecules is essentially the same. [D-Ala(3)]DPDPE crystallizes in the monoclinic space group P2(1) with cell dimensions a = 12.271(2) Angstrom, b = 9.600(a) Angstrom, c = 18.750(4) Angstrom, beta = 103.56(2)degrees, and V = 2147.2(7) Angstrom(3). The asymmetric unit contains one peptide molecule and 10 molecules of water, giving a calculated density of 1.298 g cm(-3). Comparison of these X-ray structures with the crystal structure previously reported for DPDPE indicates that there are differences in the disulfide bond region for all three peptides. ROEs determined about the disulfide regions of 1-3 in solution are indicative of a high degree of conformational interconversion, while heteronuclear coupling constants between the D-Pen(2.5) Ha and C gamma,gamma’ carbons indicate a Strong preference for a gauche (+) chi(1) angle in 2. The backbone conformations of DPDPE and [D-Ala(3)]DPDPE in the X-ray structures are virtually identical, while in [L-Ala(3)]DPDPE, there is a rotation of approximately 160 degrees about both psi(2) and phi(3) compared to DPDPE which has the effect of rotating this amide group approximately 180 degrees relative to DPDPE.