Chronic neuropathic pain may result from nerve injury and is refractory to currently available analgesic therapies. It is a debilitating pain condition and one that involves numerous complex underlying mechanisms that are poorly understood. Following nerve injury, astrocytes and microglial cells become active leading to the synthesis of neurotoxic mediators and prolonged Inducible Nitric Oxide Synthase (iNOS) transcription. Such mediators alongside excessive levels of nitric oxide (NO) have been shown to correlate with an increase in pain severity and provoke ectopic neuronal cell activity. This hypersensitivity leads to the formation of peripheral and central sensitization (1-3). We used a well established model of neuropathic pain, L5-Spinal Nerve Axotomy (L5-SNA) (4), which was carried out on Wistar rats under deep gaseous anaesthesia (2% isoflurane, 2L/min O2 and NO2). Two dosing regimens of a high selective iNOS inhibitor, N-(3-(Aminomethyl) benzyl) acetamidine dihydrochloride (1400W), or equal volume of vehicle were administered. We examined the effects of iNOS inhibition on two parameters used to characterise pain behaviour in humans; heat hyperalgesia (HH) and mechanical Allodynia (MA). Baseline behaviour was recorded prior to surgery and drug treatments and was continued for 7-14days post L5-SNA. Data are expressed as mean ±SEM. Statistical significance was calculated using either a Mann-Witney U t-test or a 2-way repeated measure ANOVA with Bonferroni’s post tests, as appropriate. Following L5-SNA and prior to drug interventions, both MA (27±0.6g to 18±0.7g, n=16) and HH (17±0.5s to 9±1s, n=16) were markedly reduced indicating a significant increase in pain levels p<0.0001). When 1400W was administered 18hr post surgery, we observed increases in both HH and MA thresholds, indicating a significant decrease in pain levels (n=9; p<0.05). The greatest significant increase in MA following 1400W was observed at 74hr post surgery (22.4±0.8g for 1400W, n=9, compared to 15.6±0.9g for vehicle, n=7; p<0.001). For HH, 162 hr post surgery, the largest increase caused by 1400W was observed (15.7±0.4s for 1400W, n=9, compared to 7.2±0.7s for vehicle, n=7; p<0.001). However, when 1400W was administered 7days post L5-SNA, no effect was observed with 1400W at any time point post-L5 SNA (n=5 per treatment group). The in vivo behavioral testing results strongly suggest that iNOS plays a key role in the hypersensitivity associated with trauma-induced NP during early stages of nerve injury. The results from this study indicate that 1400W has the ability to affect some analgesia, but became ineffective once chronic pain had fully established. We conclude that iNOS inhibiting drugs may have potential future use in neuroprotection and medical analgesia.