It is widely assumed that PI 3-kinase is not involved in contraction-stimulated glucose uptake but plays a key role in insulin-stimulated glucose transport. However, it has been difficult to develop specific evidence to document that class 1A PI 3-kinase mediate insulin-stimulated glucose uptake in skeletal muscles. PI 3-kinases are a family of 8 enzymes that are capable of phosphorylating the D-3 position of the inositol head group of phosphoinositides. Class 1A PI 3-kinase produces PtdIns(3,4,5)P3, which is believed to be the lipid signaling molecule recruiting PKB to the membrane. Global gene knockouts of the two major isoforms of PI 3-kinase (p110a and p110b) are lethal; knockin that creates a kinase dead p110a are also embryonic lethal. Data on insulin action has only been obtained from studies of heterozygous mice; these mice are insulin resistant and do have defects in insulin-induced activation of Akt/PKB but the direct effect on insulin-induced glucose transport in muscle and fat has not been reported. Indeed, overexpression of p110a or p110b induces glucose transport and GLUT-4 translocation, but overexpression of PI 3-kinases does not prove a particular PI 3-kinase isoform is involved. In particular forced overexpression of p110 causes not only large increases in PtdIns(3,4,5)P3 but also in the other D-3 inositides, so it is possible that the effects seen are due to the increase in PtdIns(3)P, PtdIns(3,4)P2 and PtdIns(3,5)P2. We have used isoform specific PI 3-kinase inhibitors against p110a, p110b, p110g and p110d to study their role in regulation of insulin-stimulated glucose uptake and PKB phosphorylation in skeletal muscles. Wortmannin and LY294002 completely blocked insulin-stimulated glucose uptake and PKB phosphorylation. However, the isoform specific PI 3-kinase inhibitors were unable to reduce glucose uptake significantly despite that PKB phosphorylation was reduced by more than 50 %. Instead, inhibition of PIKfyve, which phosphorylates the D-5 position of the inositol ring, blocked insulin-stimulated glucose uptake. Our data do not support that class 1A PI 3-kinase mediate insulin-stimulated glucose uptake in skeletal muscles, but suggest that PIKfyve is an important mediator of insulin-stimulated glucose uptake in skeletal muscles.