The present study was undertaken to examine the effect of amitriptyline on the antinociceptive effect of morphine and its underlying mechanisms in regulating glutamate transporters trafficking in morphine-tolerant rats. Long-term morphine infusion induced antinociceptive tolerance and down-regulation of glutamate transporters (GTs), GLAST, GLT-1, and EAAC1, expression in the rat spinal cord dorsal horn. Acute amitriptyline treatment potentiated morphine's antinociceptive effect, with a 5.3-fold leftward shift of morphine's dose-response curve in morphine-tolerant rats, and this was associated with GLAST and GLT-1 trafficking onto the cell surface. Similar to our previous studies, morphine challenge (10 microg/10 microl, i.t.) significant by increased the excitatory amino acids (EAAs) aspartate and glutamate level in the CSF dialysates of morphine-tolerant rats. Acute amitriptyline treatment not only suppressed this morphine-evoked EAA release, but further reduced the EAA concentration than baseline level. Furthermore, long-term morphine infusion up-regulated PKA and PKC protein expression in the spinal cord dorsal horn, while amitriptyline inhibited the increase in expression of phospho-PKA, PKCalpha, PKCbetaII, and PKCgamma. In morphine-tolerant rats, acute treatment with PKA inhibitor H89 and PKC inhibitor Gö6805 attenuated morphine tolerance and the morphine-induced CSF glutamate and aspartate elevation, and induced trafficking of GLAST and GLT-1 from cytosol onto the cell surface. These results show that acute amitriptyline treatment preserved morphine's antinociceptive effect in morphine-tolerant rats; the mechanisms may be involved in inhibition of phospho-PKA and PKC expression, and thus inducing the GLAST and GLT-1 trafficking onto glial cell surface which enhances the EAA uptake from the synaptic cleft and reduces EAA concentration in the spinal CSF.