KDN21 is a bivalent ligand that contains δ and κ opioid antagonist pharmacophores linked through a 21-atom spacer. It has been reported that KDN21 bridges δ and κ receptors that are organized as heterodimers. We have shown previously that when using [3H] diprenorphine as radioligand, KDN21 displayed greatly enhanced affinity in this series for coexpressed δ and κ opioid receptors (CDK). The present study used in vitro expression systems to investigate interactions of members of the KDN series with δ-κ heterodimers through competition binding using selective ligands and the mitogen-activated protein kinase (MAPK) assay. In this regard, the use of the selective radioligands [3H]naltrindole and [3H]norbinaltorphimine (nor-BNI) in competition binding studies revealed that KDN21 has much higher affinity than other KDN members for CDK and bound to CDK more selectively relative to mixed δ and κ opioid receptors or singly expressed δ and κ opioid receptors. Other experiments revealed that the binding of naltrindole to δ opioid receptors could increase the binding of nor-BNI to κ opioid receptors and vice versa, suggesting reciprocal allosteric modulation of receptors in the heterodimer. Regarding the selectivity of KDN21 for phenotypic δ and κ opioid receptors, we investigated the effect of KDN21 on the activation of MAPKs [extracellular signal-regulated kinases 1 and 2 (ERK1/2)] by δ- or κ-selective agonists. KDN21 inhibited the activation of ERK1/2 by [D-Pen2,D-Pen5]-enkephalin (δ1) and bremazocine (κ2) but had no effect on the activation by deltorphin II (δ2) and (+)-(5α,7α,8β)-N- methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]benzeneacetamide (U69593, κ1). 7-Benzylidenenaltrexone (δ1) and bremazocine (κ2) significantly reduced the binding of KDN21 to CDK, whereas naltriben (δ2) and U69593 produced no such change. Taken together, these data support the idea that the organization of δ and κ receptors as heterodimers gives rise to δ1 and κ2 phenotypes.