The α_2A-adrenergic receptor (AR) subtype mediates antinociception induced by the α₂AR agonists clonidine, dexmedetomidine, norepinephrine, and 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK-14,304) as well as antinociceptive synergy of UK-14,304 with opioid agonists [d-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin and deltorphin II. Differential localization of α₂-adrenergic (α_2A-, α_2B-_, α_2C-) and opioid (μ-, δ-, κ-) subtypes suggests differential involvement of subtype pairs in opioid-adrenergic analgesic synergy. The present study applies a novel imidazoline₁/α₂-adrenergic receptor analgesic, moxonidine, to test for involvement of α_2B- and α_2CARs in antinociception and antinociceptive synergy, because spinal antinociceptive activity of moxonidine shows minimal dependence on α_2AAR. Intrathecal administration of moxonidine produced similar (2–3-fold) decreases in both mutant mice with a functional knockout of α_2AAR (D79N-α_2AAR) and α_2CAR knockout (KO) mice. The potency of moxonidine was not altered in α_2BKO mice, indicating that this subtype does not participate in moxonidine-induced spinal antinociception. Moxonidine-mediated antinociception was dose dependently inhibited by the selective α₂-receptor antagonist SK&F 86466 in both D79N-α_2A mice and α_2CKO mice, indicating that α₂AR activation is required in the absence of either α_2A- or α_2CAR. Spinal administration of antisense oligodeoxynucleotides directed against the α_2CAR decreased both α_2CAR immunoreactivity and the antinociceptive potency of moxonidine. Isobolographic analysis demonstrates that moxonidine-deltorphin antinociceptive synergy is present in the D79N-α_2A mice but not in the α_2CAR-KO mice. These results confirm that the α_2CAR subtype contributes to spinal antinociception and synergy with opioids.