Glycogen synthase kinase 3 (GSK3), a key component of the insulin and wnt signaling pathways, is unusual, as it is constitutively active and is inhibited in response to upstream signals. Kinase activity is thought to be increased by intramolecular phosphorylation of a tyrosine in the activation loop (Y216 in GSK3β), whose timing and mechanism is undefined. We show that GSK3β autophosphorylates Y216 as a chaperone-dependent transitional intermediate possessing intramolecular tyrosine kinase activity and displaying different sensitivity to small-molecule inhibitors compared to mature GSK3β. After autophosphorylation, mature GSK3β is then an intermolecular serine/threonine kinase no longer requiring a chaperone. This shows that autoactivating kinases have adopted different molecular mechanisms for autophosphorylation; and for kinases such as GSK3, inhibitors that affect only the transitional intermediate would be missed in conventional drug screens.