Activation of m3 muscarinic acetylcholine receptor (mAChR), stably expressed in human embryonic kidney (HEK)-293 cells, leads to phospholipase D (PtD) stimulation, a process apparently involving Rho GTPases, as shown by studies with Clostridium botulinum C3 exoenzyme and Clostridium difficile toxin B (TcdB). Direct activation of protein kinase C (PHC) by phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), also induces PLD stimulation, which is additive to the mAChR action and which is only poorly sensitive to inactivation of Rho proteins by TcdB. To study whether Ras-like GTPases are involved in PLD regulation, we studied the effects of the TcdB variant TcdB-1470 and Clostridium sordellii lethal toxin (Test), known to inactivate Rac and some members of the Bus protein family, on PLD activities. TcdB-1470 and Test did not affect basal PLD activity and PLD stimulation by mAChR or direct G; protein activation. In contrast, PMA-induced PtD stimulation was inhibited by TcdB-1470 and Test in a time- and concentration-dependent manner, without alteration in immunologically detectable PKC isozyme levels. In membranes of HEK-293 cells pretreated with TcdB-1470 or Test, basal and stable GTP analog-stimulated PLD activities measured with exogenous phosphatidylcholine, in the presence or absence of phosphatidylinositol 4,5-bisphosphate, were not altered, In contrast, pretreatment with TcdB-1470 and Test, but not TcdB, strongly reduced PMA-stimuIated PLD activity. The addition of recombinant Rad, serving as glucosylation substrate for TcdB, Test, and TcdB-1470, did not restore PtD stimulation by PMA. Furthermore, PMA-stimulated PLD activity, suppressed by prior treatment with TcdB-1470 or Test, was not rescued by the addition of recombinant Ras (RasG12V) or Rap proteins, acting as glucosylation substrates for Test only (Das) or TcdB-1470 and Test (Rap). In contrast, the addition of recombinant Ral proteins (RalA and RalB), glucosylation substrates for TscL and TcdB-1470, but not for TcdB, to membranes of TcdB-1470- or TcsL-treated cells fully restored PtD stimulation by PMA. without altering the strict MgATP dependence of PMA-induced PLD stimulation. RalA-mediated restoration of PMA-stimulated PLD activity in membranes of Test-treated cells was not enhanced by coaddition of RasG12V. In conclusion, the data presented indicate that TcdB-1470 and Test selectively interfere with phorbol ester stimulation of PLD and suggest an essential role of Ral proteins in PKC signaling to PLD in HEK-293 cells.