To elucidate the mechanisms of phosphatidylinositol (PI) 3-kinase involvement in insulin-stimulated glucose transport activity, the epitope- tagged p110α subunit of PI 3-kinase was overexpressed in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Overexpression of p110α was confirmed by immunoblot using anti-tagged epitope antibody. p110α overexpression induced a 2.5-fold increase in PI 3-kinase activity associated with its regulatory subunits in the basal state, an increase exceeding that of the maximally insulin-stimulated control cells, while PI 3-kinase activity associated with phosphotyrosyl protein was only modestly elevated. Overexpression of p110α induced an approximately 14-fold increase in the basal glucose transport rate, which was also greater than that observed in the stimulated control. No apparent difference was observed in the cellular expression level of either GLUT1 or GLUT4 proteins between control and p110α-overexpressing 3T3-L1 adipocytes. Subcellular fractionation revealed translocation of glucose transporters from intracellular to plasma membranes in basal p110α-overexpressing cells. The translocation of GLUT4 protein to the plasma membrane was further confirmed using a membrane sheet assay. These findings indicate that an increment in PI 3-kinase activity induced by overexpression of p110α of PI 3-kinase stimulates glucose transport activity with translocation of glucose transporters, i.e., mimics the effect of insulin.