Membrane-associated phospholipase D (PLD) in HL60 cells can be activated by the small GTP-binding proteins Arf and RhoA, but polyphosphorylated inositol lipids were required for maximum activity. The intact lipid was required because neither inositol 1,4, 5-trisphosphate nor stearoyl-arachidonyl glycerol could substitute for phosphatidylinositol 4,5-bisphosphate (PIP2). Arf-stimulated but not Rho-stimulated PLD activity was increased by the inclusion of Mg2+ and ATP. ATP-dependent PLD activation occurred when phosphatidylinositol 4-phosphate (PIP), PIP2, or phosphatidylinositol 3,4,5-trisphosphate (PIP3) were included, but PIP2 formation was only detected with PIP; no PIP3 production was detected under any conditions. Therefore, the ATP-dependent increase in PLD activity cannot be explained by PIP2 or PIP3 formation. Association of endogenous Arf and RhoA with membranes was increased by incubation with GTPgammaS. This treatment increased membrane PLD and PIP kinase activities in the absence of exogenous p21 proteins. Reduction of Arf translocation suppressed the increase in PLD and PIP kinase activities, whereas complete removal of Rho but not Arf from membranes with RhoGDI was without effect on PLD activity but increased PIP kinase activity. Therefore, although recombinant Arf and Rho can activate PLD and PIP kinase in HL60 cells, it is the endogenous Arf but not Rho that regulates PLD, and thus a role for Rho in the physiological regulation of PLD in HL60 cells is unlikely.