BACKGROUND: Intrathecal (IT) administration of the α-2 adrenoceptor agonist, clonidine, produces significant analgesic effects. Although several mechanisms underlying clonidine-induced analgesia have been proposed, the possible interaction with N-methyl-D-aspartate (NMDA) receptors as a major antinociceptive mechanism has not been addressed. We designed the present study to determine whether clonidine or other analgesics can affect spinal NMDA receptor activation in rats with chronic constriction injury (CCI)-induced neuropathy. METHODS: Rats underwent unilateral CCI, and received IT clonidine (1, 5, 20 μg/rat), [d-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO, μ opioid receptor agonist, 1 μg/rat), gabapentin (anticonvulsant, 100 μg/rat) or vehicle 2 wks later. After drug injection, we measured the pain response to thermal or mechanical stimuli and used immunohistochemistry to evaluate spinal cord phosphorylated NMDA-receptor subunit 1 (pNR1) expression. RESULTS: Two weeks after CCI surgery, rats displayed significant mechanical allodynia and thermal hyperalgesia, and the spinal cord dorsal horn showed a significant increase in the number of pNR1 immunoreactive neurons. IT injection of clonidine (20 μg/rat), DAMGO and gabapentin potently reduced mechanical allodynia and thermal hyperalgesia. Importantly, IT clonidine, but not IT DAMGO or gabapentin, dose-dependently reduced CCI-induced pNR1 expression in all lamina of the spinal cord dorsal horn by 30 min after injection. In addition, IT injection of the α-2 adrenoceptor antagonist, idazoxan (40 μg/rat) 10 min before clonidine injection completely reversed clonidine's antihyperalgesic and antiallodynic effects, as well as clonidine's suppressive effect on CCI-induced NR1 phosphorylation in the spinal cord dorsal horn. CONCLUSIONS: Our data indicate that IT clonidine's antihyperalgesic/antiallodynic effect on neuropathic pain is associated with a significant reduction in spinal NMDA receptor phosphorylation and suggests a potentially novel mechanism of clonidine's action.