A challenge in opioid peptide chemistry and pharmacology is the possibility to develop novel peptides with peripheral selectivity. An enzymatically stable opioid peptide could involve an antidiarrheal effect. For this reason, we constrained the highly selective and potent tetrapeptide morphiceptin with a 6-atom bridge, resulting in a cyclic amide and an ester analogue, 2 and 3, respectively. Taking advantage of the functional coupling of the opioid receptor with the heteromultimeric G-protein-coupled inwardly rectifying K+ (GIRK1/GIRK2) channel, either the wild-type mu-, kappa-, delta- or a mutated mu-opioid receptor (hMORS329A) was functionally co-expressed with GIRK1/GIRK2 channels and a regulator of G-protein signaling (RGS4) in Xenopus laevis oocytes. The two-microelectrode voltage clamp technique was used to measure the opioid receptor activated GIRK1/GIRK2 channel responses. Both cyclic analogues were equally potent via the wild-type mu-opioid receptor hMORwt (EC(50) value 976.5 +/- 41.7 for 2 and 1017.7 +/- 60.7 for 3), while the EC(50) value for Tyr-Pro-Phe-D-Pro-NH(2) measured 59.3 +/- 4.8 nM. These three agonists displayed a four to five times decreased potency via hMORS329A as compared to the wild type. Interestingly, no effect on kappa- and delta-opioid receptors was observed. The intramolecular bridge created by cyclization of morphiceptin prevents dipeptidyl peptidase IV from interacting with these analogues. We conclude that constraining morphiceptin with a 6-atom bridge resulted in enzymatically stable peptidomimetics that are exclusively active on mu-opioid receptors. These analogues provide an interesting template in the promising approach for the design of potential antidiarrheal agents.