Abstract: Opioid receptor binding conformations for two structurally related, conformationally constrained tetrapeptides, JOM-6 (µ receptor selective) and JOM-13 (δ receptor selective), were deduced using conformational analysis of these ligands and analogs with additional conformational restrictions. Docking of these ligands in their binding conformations to opioid receptor structural models, based upon the published rhodopsin X-ray structure, implicates specific structural features of the µ and δ receptor ligand binding sites as forming the basis for the µ selectivity of JOM-6 and the δ selectivity of JOM-13. In particular, the presence of E229 in the µ receptor (in place of the corresponding D210 of the δ receptor) causes an adverse electrostatic interaction with C-terminal carboxylate-containing ligands, resulting in the observed preference of ligands with an uncharged C-terminus for the µ receptor. In addition, the requirement that the Phe3 side chain of JOM-13 assume a gauche orientation for optimal δ binding, whereas the Phe3 side chain of JOM-6 must be in a trans orientation for high-affinity µ binding can be largely attributed to the steric effect of replacement of L300 of the δ receptor by W318 of the µ receptor. Testing this hypothesis by examining the binding of JOM-6 and several of its key analogs with specific µ receptor mutants is described. Our initial results are consistent with the proposed ligand–receptor interaction models.