Phospholipase D (PLD) activity that was stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was detected in cytosol and membranes of HL60 cells. GTP gamma S-stimulated PLD activity was detected in the membranes when exogenous labeled phosphatidylcholine was used in the presence of phosphatidylethanolamine and phosphatidylinositol 4,5-bisphosphate, but not when [3H]myristic acid-labeled endogenous substrate was used. Cytosolic PLD co-chromatographed with small GTP-binding proteins on anion-exchange columns, but subsequent chromatography separated these. Reconstitution studies demonstrated ADP ribosylation factor (ARF) as a regulator of cytosolic PLD, whereas the Rho proteins RhoA and CDC42Hs were ineffective. The cytosolic enzyme showed very little activity in the absence of GTP gamma S and was stimulated by 2 mM Ca2+, whereas the membrane enzyme had significant basal activity and was inhibited by Ca2+. Rho-specific GDP dissociation inhibitor inhibited GTP gamma S stimulation of membrane PLD activity in the presence and absence of cytosol. The stimulation in GDP dissociation inhibitor-treated membranes could be partially recovered by the addition of recombinant Rho proteins (RhoA, Rac1, CDC42Hs). RhoA and Rac1 were also stimulatory in untreated membranes. However, Western blot analysis of membranes showed the presence of RhoA, but not Rac1 or CDC42Hs, suggesting that RhoA was the endogenous small GTP-binding protein involved in GTP-dependent PLD activity in membranes in the absence of cytosol. ARF also stimulated the membrane PLD in the presence of GTP gamma S, and the combination of RhoA and ARF showed a synergistic effect. These results show the presence of ARF-dependent PLD activity in both cytosol and membranes. The membranes contain another PLD activity for which the endogenous regulator appears to be RhoA. The data suggest the existence of at least two different PLD isozymes in HL60 cells.