Aims: Few studies have compared the interaction of single and repeated administration of amitriptyline (amit) with the nitrergic system and glutamatergic system in the experimental model of neuropathic pain. We aimed to evaluate theantinociceptive effectofsingleandrepeatedadministrationofamitandtoassesswhetherglutamate preceded inducible nitric oxide synthase (iNOS) inhibition as a mechanism of the analgesic effect of amit in the neuropathic model of pain. Materials and methods: Male Wistar rats were subjected to left sciatic nerve ligation. The effect of single (25mgkg−1) and repeated (10mgkg−1 daily for 3weeks) administration of amit intraperitoneally (i.p.) alone or in combination with aminoguanidine (AG i.p., 100mgkg−1 for 3days, a selective iNOS inhibitor) and MK801 (0.05mgkg−1i.p., NMDA antagonist) on resting paw posture and mechanical hyperalgesia were studied. Glutamate level and iNOS protein expression in hippocampus were detected. Key findings: Single and repeated administration of amit alone or in combination with AG or MK-801 demonstrated a significant decrease in resting pain score and increase in the pain threshold. Both glutamate and nitrite levels decreased in the hippocampi of single and repeated amit+MK-801 groups. Immunohistochemistry showed a marked decrease in iNOS immunoreactivity in rats treated with single and repeated amit+MK-801. Significance: Our results suggest that glutamate-dependent mechanisms are involved in the analgesic responses to amit administration. Importantly, glutamatergic system and its upstream nitrergic system play an important role in the antinociceptive action of amit.