Insulin stimulates glucose transport in muscle and fat cells by inducing the redistribution of a specific glucose transporter, GLUT4, from intracellular vesicles to the cell surface. Phosphoinositide (PI) 3-kinase has been implicated as a key intermediate in insulin-stimulated glucose transport by studies that have examined the effects of wortmannin and LY294002, which are thought to be specific inhibitors of this enzyme. However, the specificity of these compounds for PI 3-kinase has recently been questioned. Epidermal growth factor, which activates mitogen-activated protein kinase in mouse 3T3-L1 adipocytes, has now been shown to have no effect on PI 3-kinase activity or GLUT4 translocation in these cells. Furthermore, microinjection of a dominant negative mutant of the 85-kDa subunit of PI 3-kinase, which lacks a binding site for the catalytic 110-kDa subunit, inhibited GLUT4 translocation induced by insulin in 3T3-L1 adipocytes; microinjection of the wild-type protein had no effect. These observations indicate that PI 3-kinase is necessary for insulin-induced GLUT4 translocation and glucose transport in adipocytes.