Phospholipase D, which has been extracted from porcine brain membranes and chromatographically enriched 100-fold, was activated better by impure preparations of Arf than by purified or recombinant Arf. Examination of brain cytosol with this enriched preparation of PLD activity revealed at least three stimulatory components. One of these is Arf or the first cytoplasmic factor. A second peak of PLD-stimulating activity (cytoplasmic factor II, CFII) was resolved from Arf by anion exchange and gel filtration. This CFII can be further separated into multiple activities by chromatography with heparin-agarose. The activities were differentiated by their stimulatory properties as measured in the absence or presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) alone and in the presence of added Arf and GTP gamma S. While all of the CFII pools stimulated PLD activity to some degree and showed synergistic activation when administered in conjunction with Arf, they could be classified into two groups with distinct behavior. When used together, pools from the two respective groups showed synergistic activation of PLD. The first set of pools contained the RhoA monomeric G protein. Recombinant RhoA was used to show that it could indeed activate this enriched PLD activity and act synergistically with Arf proteins. A related monomeric G protein, Cdc42, was also effective. The second set of CFII pools were devoid of RhoA and, in contrast to the first group, demonstrated significant stimulating activity in the absence of guanine nucleotides. These data indicate that the PLD activity from brain can be modulated by several cytosolic factors and that Arf-sensitive PLD may represent a complex activity that can be regulated in an interactive fashion by a variety of cellular signaling events.