A series of side-chain constrained tyrosine derivatives, 2’,6’-dimethyl-beta-methyltyrosines (TMT), has been designed and incorporated into position 1 of the highly selective delta opioid agonists DPDPE (Tyr-D-Pen(2)-Gly-Phe-D-Pen(5)-OH) and deltorphin I (DELT I, Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2). Molecular mechanics calculations on isolated TMT residues and nuclear magnetic resonance (NMR) studies of the TMT(1)-containing peptides in DMSO showed that each of the four stereoisomers of TMT favors one particular rotamer of the side-chain chi(1) torsional angle. Therefore, substitution of four TMT isomers for Tyr(1) allows us to perform a systematic conformational scan through three staggered rotamers of the aromatic side chain, gauche(-), trans, and gauche (+), and to explore specific binding requirements of the receptor in relation to the side chain conformation. The potency and selectivity of four isomers of [TMT(1)]DPDPE and four isomers of [TMT(1)]DELT I were evaluated by radioreceptor binding assays in the rat brain using mu- and delta-selective radiolabeled ligands and by bioassays with guinea pig ileum (GPI, mu receptor) and mouse vas deferens (MVD, delta receptor). In the DPDPE series only one isomer, [(2S,3R)-TMT(1)]-DPDPE showed high potency and selectivity for the delta opioid receptors. The favorable side-chain rotamers found for this analogue, i.e., the trans rotamer of TMT(1) and the gauche (-) rotamer of Phe(4), were proposed as the most probable delta receptor-binding conformations of DPDPE analogues. Two [TMT(1)]DELT I isomers possessed considerable delta receptor potencies. The (2S,3R)-TMT(1) isomer appeared to be a superpotent, but moderately delta-selective agonist, while the (2S,3R)-TMT(1) isomer showed the highest selectivity for the delta receptors in this series. Surprisingly, [(2R,3R)TMT(1)]DELT I also was moderately potent at the delta receptor. These results suggest that the delta receptor requirements for the linear DELT I analogues may be satisfied with two different modes of binding of the (2S,3S)- and (2S,3R)-TMT(1) isomers. This study provides important guidance for the design of peptide and non-peptide ligands selective for the delta opioid receptor.