The present study was performed to evaluate the effects of the tricyclic antidepressant amitriptyline on morphine tolerance in rats. Male Wistar rats were implanted with two intrathecal (i.t.) catheters with or without a microdialysis probe, then received a continuous i.t. infusion of saline (control) or morphine (15 microg/h) and/or amitriptyline (15 microg/h) for 5 days. The results showed that amitriptyline alone did not produce an antinociceptive effect, while morphine alone induced antinociceptive tolerance and down-regulation of spinal glutamate transporters (GLAST, GLT-1, and EAAC1) in the rat spinal cord dorsal horn. Co-administration of amitriptyline with morphine attenuated morphine tolerance and up-regulated GLAST and GLT-1 expression. On day 5, morphine challenge (10 microg/10 microl) resulted in a significant increase in levels of the excitatory amino acids (EAAs), aspartate and glutamate, in CSF dialysates in morphine-tolerant rats. Amitriptyline co-infusion not only markedly suppressed this morphine-evoked EAA release, but also preserved the antinociceptive effect of acute morphine challenge at the end of infusion. Glial cells activation and increased cytokine expression (TNFalpha, IL-1beta, and IL-6) in the rat spinal cord were induced by the 5-day morphine infusion and these neuroimmune responses were also prevented by amitriptyline co-infusion. These results show that amitriptyline not only attenuates morphine tolerance, but also preserves its antinociceptive effect. The mechanisms involved may include: (a) inhibition of pro-inflammatory cytokine expression, (b) prevention of glutamate transporter down-regulation, and even up-regulation of glial GTs GLAST and GLT-1 expression, with (c) attenuation of morphine-evoked EAA release following continuous long-term morphine infusion.