OBJECTIVE: To observe the apoptosis of neural stem cells (NSCs) at differential time points after the dissociation of neurospheres by Accutase or trypsin. METHODS: The NSCs were isolated from striatum of human fetals that suffered abortion at 12-16 weeks of pregnancy. The 3(rd)-5(th) passages of NSCs were digested by Accutase or trypsin. Only vortexing was applied, and the triturating by Pasteur pipette was avoided to attenuate the injury to the cells during the dissociation. The single cells were then stained by Annexin V/propidium iodide and Hoechst 33342. The apoptosis rates 2 and 24 hours after passaging were evaluated. RESULTS: The trypan blue staining confirmed that immediately after the dissociation,the viability of cells digested by trypsin was (83.10 ± 6.76)%, which was significantly lower than that digested by Accutase,which was (91.65 ± 4.43)% (P<0.05). The apoptosis of the NSCs digested by Accutase was higher than that digested by trypsin at both 2 and 24 hours after passaging (P<0.01). Four days after the passaging, both the new clone formation rate and diameter of new spheres after trypsin digestion were significantly higher than those after Accutase digestion (P<0.01). CONCLUSIONS: Although the viability of NSCs immediately after the disassociation by trypsin is lower than that digested by Accutase, the apoptosis of NSCs subsequently caused by trypsin is lower than that caused by Accutase. Trypan blue test immediately after the disassociation can not be used as an indicator in estimating the apoptosis of NSCs during the expanding.