Previously, we found that replacement of the region around the first extracellular loop of the delta-opioid receptor (OPR) with the corresponding region of the mu-OPR gives the high affinity for [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO), a mu-opioid-selective ligand, to the resultant chimeric receptor, DMDD, suggesting that the difference in the amino acid sequence within this region between the mu- and delta-OPRs is critical for the discrimination between these receptors by DAMGO. In the current study, we carried out systematic replacements of seven non-conserved residues in this region of the delta-OPR with the corresponding amino acid found in the mu-OPR. Among the seven mutant receptors, only one mutant receptor, delta K108N, showed high affinity (Ki = 18.68 +/- 5.27 nM) for DAMGO, which was comparable to that of the DMDD receptor (Ki = 23.77 +/- 4.27 nM) and 75-fold higher than that of the wild-type delta-OPR (Ki = 1405 +/- 161 nM). Lys108 in the delta-OPR was systematically replaced with 19 kinds of amino acids other than lysine. Among the resultant mutant receptors, 14 mutants bound DAMGO with Ki values comparable to those of the DMDD receptor, ranging from 4.20 to 43.38 nM. These findings suggest that Lys108 of the delta-OPR prevents DAMGO from binding to the delta-OPR rather than that the asparagine residue at the corresponding position in the mu-OPR is necessary for DAMGO binding. In addition, the replacement of Lys108 of the delta-OPR with asparagine dramatically increased the affinity for other peptidic mu receptor-selective ligands, such as dermorphin and D-Pen-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2.