AbstractMolecular cloning has identified three opioid receptors: mu (MOR), delta (DOR) and kappa (KOR). Yet, cloning of these receptor types has offered little clarification to the diverse pharmacological profiles seen within the growing number of novel opioid ligands, which has led to the proposal of multiple subtypes. In the present study, utilizingin vitroandin vivomethods including the use of opioid receptor knockout mice, we find that certain antinociceptive effects of theKOR‐1 andKOR‐2 subtype‐selective ligands (+)‐(5α,7α,8β)‐N‐Methyl‐N‐[7‐(1‐pyrrolidinyl)‐1‐oxaspiro[4.5]dec‐8‐yl]‐benzene‐acetamide (U69, 593) and 4‐[(3,4‐Dichlorophenyl)acetyl]‐3‐(1‐pyrrolidinylmethyl)‐1‐piperazine‐carboxylic acid methyl ester fumarate (GR89, 696), respectively, are potentiated by antagonism ofMORandDORreceptors. We believe that our findings can be best explained by the existence ofKOR‐DORandKOR‐MORheteromers. We only find evidence for the existence of these heteromers in neurons mediating mechanical nociception, but not thermal nociception. These findings have important clinical ramifications as they reveal new drug targets that may provide avenues for more effective pain therapies.