Abstract: AIM: To explore the mechanism of blockade of opiate tolerance and dependence by methylene blue (MB), a soluble guanylyl cyclase (sGC) inhibitor. METHODS: An inducible nitric oxide synthase (iNOS) gene-expressing nerve cell line was used as an in vitro model system. Competitive protein binding assay and radioimmunoassay were used to examine the intracellular cAMP and cGMP content. iNOS catalytic activity was assayed by measuring the calcium-independent conversion of ³H-arginine to ³H-citrulline. Intracellular free Ca²⁺ concentration was monitored with confocal laser scanning microscopy. Cells were exposed to δ-opioid agonists DPDPE (D-Pen2,　D-Pen5-enkephalin) or morphine alone, and MB (10-6 mol.L^-1) combined with various opioid agonist for 48 hours. Cell withdrawal response was then precipitated by the addition of naloxone for 15 minutes. RESULTS: MB was found to significantly inhibit the elevation of cGMP level which resulted from long-term treatment with opioid agonists and not affect the changes of forskolin-stimulated cAMP accumulation, NOS activity and ［Ca²⁺］i. CONCLUSION: Methylene blue attenuates the development of opioid tolerance and withdrawal effects mainly through inhibition of sGC activity and subsequent down-regulation of NO-cGMP pathway.