Spinal administration of opioid and α2-adrenergic receptor (α2AR) agonists produces analgesia, and agonists interact synergistically when coadministered. The molecular mechanism underlying this synergy is largely unknown. Pharmacological studies have identified both the delta and the mu-opioid receptors (DOR and MOR) as candidate receptors capable of interacting synergistically with α2AR agonists. However, recent studies attribute the antinociceptive effect of DOR agonists to actions at the MOR, calling the role of DOR in opioid-adrenergic synergy into question. Other studies suggesting that DOR is implicated in morphine antinociception raise the possibility that DOR is nonetheless required for morphine synergy with α2AR agonists. This study aimed to determine whether DOR activation is sufficient and necessary to mediate opioid-adrenergic synergistic interactions in the spinal cord. The antinociceptive effects of clonidine, [D-Ala(2)]-deltorphin II (DeltII), morphine, and [D-Ala(2), N-Me-Phe(4), Gly-ol(5)]-enkephalin (DAMGO) were evaluated using the substance P (SP) behavioral assay in wild type (WT) and DOR-knockout (KO) mice. Opioid-adrenergic drug interactions were evaluated after spinal coadministration of clonidine with DeltII, morphine, or DAMGO. Isobolographic analyses of dose-response curves determined whether interactions were synergistic or additive. The absence of DeltII antinociceptive efficacy in DOR-KO confirmed its selectivity in the SP assay. Although DeltII+clonidine interacted synergistically in WT mice, no interaction with clonidine was observed in DOR-KO mice. Clonidine was synergistic with morphine in both mouse strains. DAMGO did not synergize with clonidine in either strain. These findings confirm that although other opioid receptors can interact synergistically with α2AR agonists, DOR is sufficient for spinal opioid-adrenergic interactions.