Insulin stimulates glucose uptake by induction of the translocation of vesicles that contain the glucose transporter Glut 4 to the plasma membrane. Phosphatidylinositol 3-kinase (PtdIns 3-kinase), which is thought to be involved in intracellular trafficking, could play a critical role in insulin-induced glucose transport. In 3T3-L1 adipocytes, insulin and platelet-derived-growth-factor (PDGF) stimulated glucose uptake by 5.8-fold and 2.4-fold, respectively, but PDGF had no significant effect on Glut 4 translocation. Nevertheless, both hormones activated PtdIns 3-kinase activity in total cell extracts. However, insulin and PDGF had different effects on the stimulation of PtdIns 3-kinase activity in several subcellular fractions, and the movements of insulin-receptor substrate (IRS) 1 and the p85 subunit of PtdIns 3-kinase between subcellular compartments. PDGF stimulated PtdIns 3-kinase activity almost exclusively in the plasma membrane, and induced translocation of the p85 subunit from the cytosol to the plasma membrane, where the PDGF receptor was phosphorylated on tyrosine residues. In contrast, insulin stimulated PtdIns 3-kinase activity in the plasma membrane, in low-density microsomes (LDM) and in cytosol. Furthermore, insulin induced the translocation of p85 from the cytosol to LDM and the translocation of IRS 1 from LDM to the cytosol. These data indicate that insulin and PDGF have different effects on the activation of PtdIns 3-kinase and on the movement of IRS 1 and PtdIns 3-kinase between subcellular compartments. We would like to suggest that a crucial event in the stimulation of glucose uptake by insulin could be that insulin, but not PDGF, induces activation of PtdIns 3-kinase in the cytosol and in LDM, the compartment enriched in Glut-4-containing vesicles.