Glycogen synthase kinase (GSK)-3 was identified initially as an enzyme that regulates glycogen synthesis in response to insulin, but more recent studies indicate that it is also involved in numerous cellular processes, including cell survival, cell cycle regulation, proliferation, and differentiation. Because extracellular ATP exerts trophic actions on astrocytes, we investigated a possible signaling linkage from P2 purinergic receptors to GSK3β. Addition of ATP to primary cultures of rat cortical astrocytes resulted in phosphorylation of Ser9 on GSK3β and a concomitant decrease in GSK3 activity. UTP and 2′,3′-O-(4-benzoyl)-benzoyl ATP (BzATP) increased phosphorylation of Ser9 on GSK3β indicating that metabotropic P2Y and ionotropic P2X receptors are coupled to GSK3β. Signaling studies showed that phosphorylation of Ser9-GSK3β in response to ATP was inhibited by downregulation of protein kinase C (PKC) but not by blockade of Akt or p70 S6 kinase pathways. PKC also links P2 receptors to ERK in astrocytes, but inhibition of ERK signaling did not block phosphorylation of Ser9-GSK3β stimulated by P2 receptors. Mechanical strain, which releases ATP, also stimulated Ser9 phosphorylation and this was attenuated by hydrolysis of extracellular ATP with apyrase or by blockade of P2 receptors. We conclude that P2 receptors are coupled to GSK3β by a PKC-dependent pathway that is independent of Akt, p70 S6 kinase, and ERK pathways. These findings suggest that purinergic signaling contributes to the regulation of GSK3β functions, one of which may be the response of astrocytes to CNS injury on release of ATP. © 2006 Wiley-Liss, Inc.