The dominant negative p85alpha regulatory subunit (delta p85alpha) of phosphatidylinositol (PI) 3-kinase or dominant negative Ras (N17Ras) was overexpressed in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Functional expression of delta p85alpha and N17Ras was confirmed by marked inhibition of insulin-stimulated PI 3-kinase activity and mitogen-activated protein kinase activity, respectively. N17Ras expression did not affect glucose transport activity, whereas delta p85alpha expression inhibited insulin-stimulated glucose transport with impairment of GLUT-4 translocation, although inhibition of glucose transport activity was less remarkable than that of PI 3-kinase activity in delta p85alpha-expressing cells. Thus the Ras signaling pathway does not play a major part in either translocation or intrinsic activity of glucose transporters, but PI 3-kinase activation, via phosphotyrosyl proteins and heterodimeric PI 3-kinase, plays a pivotal role in insulin-stimulated glucose transport. However, a discrepancy was observed between PI 3-kinase activity and glucose transport activity, suggesting a possibility that a different pathway(s) is involved in insulin-stimulated intrinsic activity of glucose transporters.