The structural basis of μ-opioid receptor (OPR) for the specificity in its ligand binding was investigated using chimeric μ/δ-OPRs. Replacement of the region around the first extracellular loop of δ-OPR with the corresponding region of μ-OPR gave the resultant chimeric receptor the similar affinity to DAMGO compared with the native μ-OPR. The reciprocal replacement deprived the high affinity to DAMGO from μ-OPR. These results indicate that the difference(s) in the structure around the first extracellular loop is critical for DAMGO to distinguish between μ- and δ-OPRs. Furthermore, displacement studies revealed that this region is partly involved in the discrimination between μ- and δ-OPRs by other peptidic μ-selective ligands, such as dermorphin, morphiceptin and CTOP, but not by non-peptidic ligands, such as morphine and naloxone.