Eukaryotic initiation factor eIF2B catalyses a key regulatory step in mRNA translation, eIF2B and total protein synthesis are acutely activated by insulin, and this requires phosphatidylinositol 3-kinase (PI 3-kinase). The epsilon-subunit of eIF2B is phosphorylated by glycogen synthase kinase-3 (GSK-3), which is inactivated-by insulin in a PI 3-kinase-dependent manner, Here we identify the phosphorylation site in eIF2B epsilon as Ser(540) and show that treatment of eIF2B with GSK-3 inhibits its activity. Ser(540) is phosphorylated in intact cells and undergoes dephosphorylation in response to insulin, This is blocked by PI 3-kinase inhibitors, Insulin-induced dephosphorylation of this inhibitory site in eIF2B seems likely to be important in the overall activation of translation by this hormone.