Toward developing new potential analgesics, this first structure-activity relationship study of opiorphin (H-Gln-Arg-Phe-Ser-Arg-OH), a human peptide inhibiting enkephalin degradation, was performed. A systematic Ala scanning proved that Phe(3) is a key residue for neprilysin and aminopeptidase N (AP-N) ectoenkephalinase inhibition. A series of Phe(3)-halogenated analogues revealed that halogen bonding based optimization strategies are not applicable to this residue. Additional substituted Phe(3) derivatives showed that replacing l-Phe(3) for d-Phe(3) increased the AP-N inhibition potency by 1 order of magnitude. NMR studies and molecular mechanics calculations indicated that the improved potency may be due to CH-π stacking interactions between the aromatic ring of d-Phe(3) and the Hγ protons of Arg(2). This structural motif is not possible for the native opiorphin and may be useful for the design of further potent and metabolically stable analogues.