Retroviral Gag proteins direct virus particle assembly from the plasma membrane (PM). Phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] plays a role in PM targeting of several retroviral Gag proteins. Here we report that depletion of intracellular PI(4,5)P(2) and phosphatidylinositol-(3,4,5)-triphosphate [PI(3,4,5)P(3)] levels impaired Rous sarcoma virus (RSV) Gag PM localization. Gag mutants deficient in nuclear trafficking were less sensitive to reduction of intracellular PI(4,5)P(2) and PI(3,4,5)P(3), suggesting a possible connection between Gag nuclear trafficking and phosphoinositide-dependent PM targeting.