Homo- and heterodimerization of the opioid receptors with functional consequences were reported previously. However, the exact nature of these putative dimers has not been identified. In current studies, the nature of the heterodimers was investigated by producing the phenotypes of the 1:1 heterodimers formed between the constitutively expressed μ-opioid receptor (MOR) and the ponasterone A-induced expression of δ-opioid receptor (DOR) in EcR293 cells. By examining the trafficking of the cell surface-located MOR and DOR, we determined that these two receptors endocytosed independently. Using cell surface expression-deficient mutants of MOR and DOR, we observed that the corresponding wild types of these receptors could not rescue the cell surface expression of the mutants, whereas the antagonist naloxone could. Furthermore, studies with constitutive or agonist-induced receptor internalization also indicated that MOR and DOR endocytosed independently and could not "drag in" the corresponding wild types or endocytosis-deficient mutants. Additionally, the heterodimer phenotypes could be eliminated by the pretreatment of the EcR293 cells with pertussis toxin and could be modulated by the deletion of the RRITR sequence in the third intracellular loop that is involved in the receptor-G protein interaction and activation. These data suggest that MOR and DOR heterodimerize only at the cell surface and that the oligomers of opioid receptors and heterotrimeric G protein are the bases for the observed MOR-DOR heterodimer phenotypes.