We previously showed that intrathecal co-administration of amitriptyline with morphine upregulates the expression of the glial glutamate transporters glutamate-aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) and restores neuronal glutamate transporter excitatory amino acid carrier 1 (EAAC1) expression in chronically morphine-infused rats. The present study examined the role of nuclear transcription factor-kappaB (NF-kappaB) in the regulation of the expression of GLAST, GLT-1, and EAAC1 following long-term amitriptyline/morphine co-infusion. Male Wistar rats were implanted with two intrathecal catheters with or without a microdialysis probe; one of the catheters was used for continuous infusion of saline (control), morphine (15 microg/h), or morphine plus amitriptyline (both 15 microg/h) for 5 days, while the other was used for a single daily intrathecal injection of the NF-kappaB inhibitor Ro106-9920 (10 microl of 10 microM) for 5 days. We found that amitriptyline co-infusion restored the antinociceptive effect of morphine (4.5-fold right-shift in the morphine dose-response curve compared with a 65-fold right-shift in its absence) and this effect was inhibited by Ro106-9920 administration (48-fold right-shift). Moreover, amitriptyline/morphine co-infusion increased IkappaBalpha phosphorylation and the translocation of NF-kappaB p65 from the cytosol to the nucleus. Daily intrathecal injection of Ro106-9920 prevented the amitriptyline/morphine-induced NF-kappaB p65 translocation and reversed the amitriptyline/morphine-induced GLAST and GLT-1 upregulation and inhibited the restoration of EAAC1 expression. The Ro106-9920 injections abolished the inhibitory effect of amitriptyline on the morphine-evoked release of excitatory amino acids into the spinal cerebrospinal fluid (CSF) dialysates. In conclusion, amitriptyline/morphine co-infusion restores the antinociceptive effect of morphine and upregulates GLAST and GLT-1 expression and restores EAAC1 expression to baseline levels, thus reducing excitatory amino acid levels in the spinal CSF dialysates. The mechanism involves activation of the NF-kappaB pathway, but may also involve other pathways.