Recent studies have revealed a positive correlation between astrocyte apoptosis and rapid disease progression in persons with neurodegenerative diseases. Glycogen synthase kinase 3β (GSK-3β) is a molecular regulator of cell fate in the central nervous system and a target of the phosphatidylinositol 3-kinase (PI-3K) pathway. We have therefore examined the role of the PI-3K pathway, and of GSK-3β, in regulating astrocyte survival. Our studies indicate that inhibition of PI-3K leads to apoptosis in primary cortical astrocytes. Furthermore, overexpression of a constitutively active GSK-3β mutant (S9A) is sufficient to cause astrocyte apoptosis, whereas an enzymatically inactive GSK-3β mutant (K85M) has no effect. In light of reports on the interplay between GSK-3β and nuclear factor κB (NF-κB), and because of the antiapoptotic activity of NF-κB, we examined the effect of GSK-3β overexpression on NF-κB activation. These experiments revealed strong inhibition of NF-κB activation in astrocytes upon overexpression of the S9A, but not the K85M, mutant of GSK-3β. This was accompanied by stabilization of the NF-κB-inhibitory protein, IκBα and down-regulation of IκB kinase (IKK) activity. These findings therefore implicate GSK-3β as a regulator of NF-κB activation in astrocytes and suggest that the pro-apoptotic effects of GSK-3β may be mediated at least in part through the inhibition of NF-κB pathway.