Given the high homology in amino acid sequence between the delta-opioid receptor and the two other types (mu and kappa), distinct residues in this receptor may confer its selectivity to some ligands. In order to identify molecular determinants in the human delta receptor responsible for the selectivity of delta-selective ligands, two different delta/mu chimeras were constructed. In the first one, the delta sequence from the top of transmembrane 5 to the C terminus was replaced by the equivalent mu sequence, and in the second one, 13 consecutive residues in the third extracellular loop region of the delta receptor were replaced by the mu counterpart. These two chimeras retained the ability to bind the nonselective bremazocine but completely lost the ability to bind different delta-selective ligands. These results suggested that the region of the third extracellular loop of the delta receptor is crucial for the type selectivity. Furthermore, an alanine scan was performed by site-directed mutagenesis of 20 amino acids located in or proximal to the third extracellular loop. Among all the point mutations, only mutations of Trp-284, Val-296, or Val-297 significantly decreased the binding of delta-selective ligands tested. Moreover, combined mutation of Trp-284, Val-296, and Val-297 considerably decreased the affinities of the receptor for delta-selective ligands compared with the single point mutations. These findings suggest that Trp-284, Val-296, and Val-297 are crucial residues involved in the delta receptor type selectivity.