Endomorphin-1 is a novel endogenous peptide that is highly selective for the mu-opioid receptor over the delta- and kappa-opioid receptors. The structural basis of high selectivity of endomorphin-1 to the mu-opioid receptor was examined using chimeric receptors between mu- and delta-opioid receptors and those between mu- and kappa-opioid receptors. The chimeric receptors were constructed by using restriction enzyme sites intrinsically possessed by or introduced to the mu-, delta- and kappa-opioid receptor cDNAs. The junctions for the construction were located at the first intracellular loop (Bbs I site), third transmembrane domain (Afl III site) and fifth transmembrane domain (Bgl II site). The competitive binding assay using chimeric receptors revealed that the region from the Bbs I site to the Afl III site, including the first extracellular loop, contributes to the discrimination between mu- and delta-opioid receptors by endomorphin-1 more than any other regions. However, the region from the Afl III site to the Bgl II site and that from the Bgl II site to the carboxy terminal also somewhat contribute to the discrimination between mu- and delta-opioid receptors. For the discrimination between mu- and kappa-opioid receptors, two regions, that is, the region from the Bbs I site to the Afl III site and that from the Bgl II site to the carboxy terminal, were shown to be important. The present results show that endomorphin-1 discriminates the mu-opioid receptor from the other two types of opioid receptors by recognizing the differences in several amino acid residues widely distributed through the receptor structure. We previously reported that DAMGO, a synthetic highly mu-selective peptide, discriminates between mu- and delta-opioid receptors by recognizing the difference in only one amino acid residue and discriminates between mu- and kappa-opioid receptors by recognizing the difference in four residues localized in the restricted region. Although both endomorphin-1 and DAMGO are mu-opioid receptor selective peptides, molecular mechanisms for mu-selectivity are different between these peptides.