The heptadecapeptide orphanin FQ (OFQ) is an endogenous ligand to an opioid-like G protein-coupled receptor. Although the primary structure of OFQ exhibits some similarity to the opioid peptides, OFQ is not recognized by opioid receptors nor does the OFQ receptor bind opioid ligands. In order to investigate the structural determinants of this ligand/receptor selectivity, we conducted a systematic structure-activity study on OFQ to characterize which sites of the molecule are important for receptor activation. Alanine- and D-amino acid-scanning mutagenesis revealed several residues in the amino-terminal half of OFQ which participate in both receptor binding and activation. Most strikingly, the Phe1 position could be changed to a tyrosine without loss of biological activity. In addition, the OFQ receptor seemed to require recognition of the complete peptide molecule for activation. These results indicate that the mode of interaction of OFQ with its receptor may be different from that of the opioid peptides with their respective receptors and might therefore account for the observed selectivity.