Insulin stimulation of 3T3-L1 adipocytes causes rapid translocation of actin and the GLUT4 glucose transporter to the plasma membrane, Both processes depend on the activity of phosphatidylinositol 3-kinase, Using single cell microinjection, we have transiently expressed a constitutively activated mutant of phosphatidylinositol 3-kinase, p110*, in 3T3-L1 adipocytes, Fluorescent detection of GLUT4 protein and actin within these cells demonstrates that expression of p110* is sufficient to cause translocation of GLUT4 to the plasma membrane and the formation of actin membrane ruffles, These effects are inhibited by wortmannin in the p110*-expressing cells, indicating that the phosphatidylinositol 3-kinase activity of the protein is required, Overexpression of an identical protein containing a point mutation in the kinase domain, p110*Delta kin, was incapable of mediating either action, confirming that neither the microinjection process nor a nonspecific effect of the protein was responsible for the observed effects, These data suggest that although insulin is capable of inducing numerous signaling pathways, the isolated activation of phosphatidylinositol 3-kinase can initiate the signaling cascade leading to both actin rearrangement and GLUT4 translocation in the absence of insulin stimulation.