Phosphorylation of the endogenous GSK3α (glycogen synthase kinase-3α) at Tyr²⁷⁹ and GSK3β at Tyr²¹⁶ was suppressed in HEK-293 or SH-SY5Y cells by incubation with pharmacological inhibitors of GSK3, but not by an Src-family inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), or a general protein tyrosine kinase inhibitor (genistein). GSK3β transfected into HEK-293 cells or Escherichia coli became phosphorylated at Tyr²¹⁶, but catalytically inactive mutants did not. GSK3β expressed in insect Sf 21 cells or E. coli was extensively phosphorylated at Tyr²¹⁶, but the few molecules lacking phosphate at this position could autophosphorylate at Tyr²¹⁶in vitro after incubation with MgATP. The rate of autophosphorylation was unaffected by dilution and was suppressed by the GSK3 inhibitor kenpaullone. Wild-type GSK3β was unable to catalyse the tyrosine phosphorylation of catalytically inactive GSK3β lacking phosphate at Tyr²¹⁶. Our results indicate that the tyrosine phosphorylation of GSK3 is an intramolecular autophosphorylation event in the cells that we have studied and that this modification enhances the stability of the enzyme.