Over-production of nitric oxide is pathogenic for neuronal apoptosis around the ischemic area following ischemic brain injury.In this study,an apoptotic model in rat hippocampal neurons was established by 0.5 mmol/L 3-morpholinosyndnomine(SIN-1),a nitric oxide donor.The models were then cultured with 0.1 mmol/L of 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid(DIDS;the chloride channel blocker) for 18 hours.Neuronal survival was detected using the 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay,and apoptosis was assayed by Hoechst 33342-labeled neuronal DNA fluorescence staining.Western blot analysis and immunochemiluminescence staining were applied to determine the changes of activated caspase-3 and CIC-3 channel proteins.Real-time PCR was used to detect the mRNA expression of CIC-3.The results showed that SIN-1 reduced the neuronal survival rate,induced neuronal apoptosis,and promoted ClC-3 chloride channel protein and mRNA expression in the apoptotic neurons.DIDS reversed the effect of SIN-1.Our findings indicate that the increased activities of the ClC-3 chloride channel may be involved in hippocampal neuronal apoptosis induced by nitric oxide. Over-production of nitric oxide is pathogenic for neuronal apoptosis around the ischemic area following ischemic brain injury. In this study, an apoptotic model in rat hippocampal neurons was established by 0.5 mmol / L 3-morpholinosyndnomine (SIN-1), a nitric oxide donor. These models were then cultured with 0.1 mmol / L of 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid (DIDS; the chloride channel blocker) for 18 hours. Neuronal survival was detected using the 3- (4, 5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay, and apoptosis was assayed by Hoechst 33342-labeled neuronal DNA fluorescence staining. Western blot analysis and immunochemiluminescence fluorescence staining were applied to determine the changes of activated caspase- 3 and CIC-3 channel proteins. Real-time PCR was used to detect the mRNA expression of CIC-3.The results showed that SIN-1 reduced the neuronal survival rate, induced neuronal apoptosis, and promoted ClC-3 chloride channel protein and mRNA expression in the apoptotic neuro ns.DTV reversed the effect of SIN-1.Our findings indicate that the increased activities of the ClC-3 chloride channel may be involved in hippocampal neuronal apoptosis induced by nitric oxide.