Stimulation of glucose transport is among the most important metabolic actions of insulin. Studies in adipose cells have demonstrated that insulin stimulates its receptor to phosphorylate tyrosine residues in IRS-1, leading to activation of phosphatidylinositol 3-kinase, which plays a necessary role in mediating the translocation of the insulin-responsive glucose transporter GLUT4 to the cell surface. Akt is a serine-threonine kinase recently identified as a direct downstream target of phosphatidylinositol 3-kinase. A previous study in 3T3-L1 cells showed that overexpression of a constitutively active mutant of Akt is sufficient to recruit GLUT4 to the cell surface. Since effects of overexpression of signaling molecules in tissue culture models do not always reflect physiological function, we have overexpressed a dominant inhibitory mutant of Akt in rat adipose cells to investigate the effects of inhibiting endogenous Akt in a physiologically relevant insulin target cell. Cells were transfected with either wild type (Akt-WT), constitutively active (Akt-myr), or dominant inhibitory (Akt-K179A) forms of Akt, and effects of overexpression of these constructs on insulin-stimulated translocation of a cotransfected epitope-tagged GLUT4 were studied. Overexpression of Akt-WT resulted in significant translocation of GLUT4 to the cell surface even in the absence of insulin. Interestingly, overexpression of Akt-myr resulted in an even larger effect that was independent of insulin. More importantly, overexpression of Akt-K179A (kinase-inactive mutant) significantly inhibited insulin-stimulated translocation of GLUT4. Taken together, our data suggest that Akt is not only capable of stimulating the translocation of GLUT4 but that endogenous Akt is likely to play a significant physiological role in insulin-stimulated glucose uptake in insulin targets such as muscle and adipose tissue.