Phospholipase D (PLD) is believed to play an important role in cell signal transduction: PLD catalyzes the hydrolysis primarily of phosphatidylcholine (PC) to produce phosphatidic acid that may serve as a lipid second messenger. Although the mechanism of PLD activation has not yet been fully understood, a member of the low molecular weight GTP-binding protein (small G protein) superfamily, ADP-ribosylation factor (ARF), has been identified as a PLD-activating factor. In addition to ARF, we found that RhoA, another member of the small G proteins, activated rat brain PLD, and that ARF and RhoA synergistically stimulated the enzyme activity. When proteins of bovine brain cytosol were subjected to anion exchange column chromatography and then reconstituted with rat brain PLD partially purified from the membranes, fractions eluted at 60 mM NaCl, where ARF was not detected, activated the enzyme in a guanosine 5'-O-(3-thiotriphosphate)-dependent manner. This PLD-stimulating activity seemed to be attributed to a small G protein RhoA. Evidence provided includes the findings that: (1) the partially purified preparation of the PLD-activating factor by subsequent column chromatographies contained a 22 kDa substrate for botulinum C3 exoenzyme ADP-ribosyltransferase; (2) the 22 kDa protein strongly reacted with anti-RhoA antibody; (3) the treatment of the partially purified PLD-activating factor with C3 exoenzyme and NAD together, but not individually, significantly inhibited the PLD-stimulating activity; and (4) recombinant isoprenylated RhoA activated the PLD. On the contrary, recombinant nonisoprenylated RhoA failed to activate the PLD. Interestingly, the partially purified PLD-activating factor and ARF synergistically activated rat brain PLD, and recombinant isoprenylated RhoA could substitute for the partially purified preparation. These results conclude that rat brain PLD is regulated by RhoA in concert with ARF, and that the post-translational modification of RhoA is essential for its function as the PLD activator.