To elucidate which portions of the opioid receptor molecules are involved in the ligand selectivity, we have expressed chimeric receptors between the rat delta- and mu-opioid receptors from cDNAs and analyzed their ligand binding properties, We demonstrate that the major binding determinant for the delta-selective enkephalin related peptide, [D-Pen(2),D-Pen(5)]enkephalin, resides within the region comprising the transmembrane segments V-VII and the intervening loop regions, On the other hand, the region spanning from the intracellular loop I to the amino terminal half of the transmembrane segment III is shown to be involved in determining high-affinity binding of the mu-selective enkephalin-related peptides, [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin and [D-Ala(2), MePhe(4),Met-ol(5)]enkephalin, whereas the major determinant nant for binding of the mu-selective alkaloids, morphine and codeine, is demonstrated to exist in the region spanning the transmembrane segments V-VII, These results indicate that distinct regions of the opioid receptor determine the selectivity for the delta- and the mu-selective enkephalin-related peptides and that the binding determinant for the mu-selective alkaloids is distinct from that for the mu-selective enkephalin-related peptides.