The exposure of 3T3-L1 fibroblasts to growth factors results in a 2-to-3-fold increase in 2-deoxyglucose transport and a approximately 50% to 80% increase in cell-surface transferrin receptor levels. We sought to determine the role of phosphatidylinositol-3'-kinase and p70 ribosomal S6 kinase in these stimulations, using selective inhibitors of these enzymes. Both basal and growth factor-stimulated deoxyglucose transport are blocked by wortmannin, but with different IC50 values (65 nM vs. 15 nM, respectively), suggesting a functional difference between these two states. This is accompanied by the accumulation of glucose transporters in intracellular locations. Both basal and growth factor-stimulated cell-surface transferrin receptor levels are downregulated by wortmannin, but with identical IC50 values (approximately 15 nM). These two proteins are known to recycle between an intracellular site and the plasma membrane in these cells, thus implying a functional role for phosphatidylinositol-3'-kinase in membrane recycling. In an effort to determine whether the effect of wortmannin was selective for the protein component of this recycling, we examined fluid-phase endocytosis of radiolabeled mannitol. Wortmannin was without effect on the fluid phase accumulation of mannitol, suggesting that the effects on membrane traffic are limited to the protein component of recycling membranes. Rapamycin, an inhibitor of p70 ribosomal S6 kinase, was without effect on any of these parameters, but both rapamycin and wortmannin inhibit growth factor-stimulated p70 ribosomal S6 kinase activity. These data support an important role for phosphatidylinositol-3'-kinase, but not p70 ribosomal S6 kinase, in the regulation of membrane protein traffic. We suggest that this enzyme may be involved in sorting of membrane proteins during trafficking.