Abstract: AIM　To determine the effect of chronic treatment with opioid agonists on NO-cGMP signal system on the basis of successful establishment of a NG-LNCXiNOS cell line expressing iNOS cDNA and a cell model of opioid tolerance and naloxone-precipitated withdrawal. METHODS　NOS activity and cGMP content were determined by the conversion of 3H-Arginine to 3H-Citrulline and radioimmunoassay, respectively. Western blot analysis and NADPH diaphorase (NADPH-d) histochemical assay were used to detecte the level of iNOS gene expression and NADPH-d activity which is a histochemical marker for NOS. RESULTS　Long-term exposure of NG-LNCXiNOS cells to various opioid agonists enhanced the cytosolic iNOS activity, accompanying the increase in intracellular cGMP content in a dose-dependent manner. The order of potencies was DPDPE>DADLE> morphine. The EC₅₀ values of the above indicators were nmol.L^-1 level. When naloxone induced cell withdrawal, the iNOS activity and cGMP level were dramatically higher than those with agonists alone. Pretreatment of the cells with the more efficacious δ-ligand (DPDPE) for 48 hours also may lead to high-level expression of iNOS protein and elevate the number of NADPH diaphorase-positive cells. CONCLUSION　Chronic opioid treatment was shown to up-regulate the NO-cGMP signal pathway, which may reflect an important biochemical change accounting for development of tolerance to and dependence on opiate. Thus, NG-LNCXiNOS cells provide a suitable system for studying the relationship between AC-cAMP and NO-cGMP signal system on the molecular mechanisms of opiate tolerance and dependence.