Inorganic phosphate (Pi) is a major regulator of cell metabolism. The Pi transport activity in the plasma membrane is a main determinant of the intracellular level of this ion. In bone-forming cells, Pi transport is important for the calcification of the bone matrix. In this study, the effect of platelet-derived growth factor (PDGF) on Pi transport activity and the signaling mechanism involved in this cellular response were analyzed. The results indicate that PDGF is a potent and selective stimulator of sodium-dependent Pi transport in the mouse calvaria-derived MC3T3-E1 osteoblast-like cells. The change in Pi transport induced by PDGF-BB was dependent on translational processes and affected the Vmax of the Pi transport system. These observations suggested that enhanced Pi transport activity in response to PDGF resulted from insertion of newly synthesized Pi transporters in the plasma membrane. The role of activation of mitogen activated protein (MAP) kinase, phospholipase C (PLC)γ or phosphatidylinositol 3-kinase (PI-3–kinase), in mediating this effect of PDGF, was investigated. A selective inhibitor of the PDGF receptor tyrosine kinase activity (CGP 53716) completely blocked PDGF-induced protein tyrosine phosphorylation of several proteins including the PDGF receptor, PLCγ, MAP kinase, and association of the p85 subunit of PI-3′-kinase. Associated with this effect, the increase in Pi transport induced by PDGF was completely blunted by 5 μM CGP 53716. Inhibition of MAP kinase activity by cAMP agonists did not influence Pi transport stimulation induced by PDGF. However, inhibitors of protein kinase C completely blocked this response. A selective inhibitor of PI-3-kinase, LY294002, also significantly reduced this effect of PDGF. In summary, these results indicate that PDGF is a potent and selective stimulator of Pi transport in osteoblastic cells. The mechanism responsible for this effect is not mediated by MAP kinase but involves tyrosine phosphorylation-dependent activation of PLCγ and PI-3-kinase.