Stimulation of phospholipase D by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in rat liver plasma membranes indicates the involvement of GTP-binding proteins. We used RhoGDI, an inhibitor of GDP dissociation from small GTP-binding proteins of the Rho family, to determine the involvement of these proteins. Incubation, and subsequent washing, of plasma membranes with RhoGDI dose-dependently diminished GTP gamma S-stimulated phospholipase D activity, as determined by accumulation of phosphatidylethanol in the presence of ethanol. Incubation with RhoGDI also caused a rapid and dose-dependent appearance of RhoA in the wash, which was associated with the inhibition of phospholipase D. RhoGDI also rapidly extracted Cdc42 from membranes, but Rac1 was not extracted. Full reconstitution of GTP gamma S-stimulated phospholipase D in RhoGDI-washed membranes was achieved with recombinant RhoA. There was partial reconstitution with Rac1 and no enhancement with Cdc42 or ADP-ribosylation factor. The response to RhoA was dose-dependent (EC50 = 0.5 microM). ADP-ribosylation of RhoA by Clostridium botulinum C3 exoenzyme did not affect its ability to recover GTP gamma S-stimulated phospholipase D activity in RhoGDI-washed membranes. These findings support a role for GTP-binding proteins of the Rho family in the activation of membrane-associated phospholipase D and implicate RhoA as the major protein involved.