Abstract.Aims/hypothesis: PI(3,4,5)P3 produced by PI3-kinase seems to be a key mediator for insulins metabolic actions. We have recently cloned rat SHIP2 cDNA which is abundantly expressed in target tissues of insulin. Here, we clarify the role of SHIP2 possessing 5-phosphatase activity toward PI(3,4,5)P3 in insulin signalling in the skeletal muscle. Methods: The role of SHIP2 in insulin-induced glycogen synthesis was studied by expressing wild-type (WT)-SHIP2 and a 5-phosphatase defective (��IP)-SHIP2 into L6 myotubes by means of adenovirus mediated gene transfer. Results: The early events of insulin signalling including tyrosine phosphorylation of the insulin receptor and IRS-1, IRS-1 association with the p85 subunit, and PI3-kinase activity were not affected by expression of WT- and ��IP-SHIP2. Although PI(3,4,5)P3 and PI(3,4)P2 are known to possibly activate a downstream molecule of PI3-kinase Akt in vitro, overexpression of WT-SHIP2 inhibited insulin-induced phosphorylation and activation of Akt. Conversely, Akt activity was increased by expression of ��IP-SHIP2. GSK3 located downstream of Akt is an important molecule to further transmit insulin signal for glycogen synthesis in skeletal muscles. In accordance with the results of Akt, insulin-induced phosphorylation and inactivation of GSK3, subsequent activation of glycogen synthase and glycogen synthesis were decreased by expression of WT-SHIP2, whereas these events were increased by expression of ��IP-SHIP2. Conclusion/interpretation: Our results indicate that SHIP2 plays a negative regulatory role via the 5-phosphatase activity in insulin signalling, and that PI(3,4,5)P3 rather than PI(3,4)P2 is important for in vivo regulation of insulin-induced Akt activation leading to glycogen synthesis in L6 myotubes. [Diabetologia (2001) 44: 12581267]