Abstract: A series of cyclic, disulfide- or dithioether-containing tetrapeptides based on previously reported potent μ- and δ-selective analogs has been explored with the aim of improving their poor affinity to the κ-opioid receptor. Specifically targeted were modifications of tetrapeptide residues 3 and 4, as they presumably interact with residues from transmembrane helices 6 and 7 and extracellular loop 3 that differ among the three receptors. Accordingly, tetrapeptides were synthesized with Phe3 replaced by aliphatic (Gly, Ala, Aib, Cha), basic (Lys, Arg, homo-Arg), or aromatic sides chains (Trp, Tyr, p-NH2Phe), and with d-Pen4 replaced by d-Cys4, and binding affinities to stably expressed μ-, δ-, and κ-receptors were determined. In general, the resulting analogs failed to exhibit appreciable affinity for the κ-receptor, with the exception of the tetrapeptide Tyr-c[d-Cys-Phe-d-Cys]-NH2, cyclized via a disulfide bond, which demonstrated high binding affinity toward all opioid receptors (Kiμ = 1.26 nm, Kiδ = 16.1 nm, Kiκ = 38.7 nm). Modeling of the κ-receptor/ligand complex in the active state reveals that the receptor-binding pocket for residues 3 and 4 of the tetrapeptide ligands is smaller than that in the μ-receptor and requires, for optimal fit, that the tripeptide cycle of the ligand assume a higher energy conformation. The magnitude of this energy penalty depends on the nature of the fourth residue of the peptide (d-Pen or d-Cys) and correlates well with the observed κ-receptor binding affinity.