Integrin-mediated adhesion is known to stimulate production of phosphatidylinositol4, 5-bisphosphate(4, 5-PIP*) and increase 4, 5-PIP2 hydrolysis in response to platelet-derived growth factor (PDGF). We now show that treatment of cells with lovastatin, which inhibits modification of small GTP-binding proteins, reduced PIP2 levels and decreased calcium mobilization in response to PDGF and thrombin. In ceil lysates, GTPyS stimulated PIP 5-kinase activity, and this effect was blocked by botulinum C3 exoenzyme, suggesting that Rho was responsible. GTP-bound recombinant Rho stimulated PIP 5-kinase activity, whereas GDP-Rho was much less potent and GTP-bound Rat was ineffective. Microinjected botulinum C3 exoenzyme caused diminished calcium mobilization in response to PDGF or thrombin. Conversely, microinjection of activated Rho reversed the decrease in calcium mobilization normally seen in nonadherent cells. These data demonstrate that Rho regulates 4, 5-PIP2 synthesis and, indirectly, 4, 5-PIP2 hydrolysis. They also raise the possibility that PIP2 synthesis could mediate the effects of Rho on the actin cytoskeleton.