Rho proteins have been reported to activate phospholipase D (PLD) in in vitro preparations. To examine the role of Rho proteins in receptor signaling to PLD, we studied the effect of Clostridium difficile toxin B, which glucosylates Rho proteins, on the regulation of PLD activity in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor (mAChR). Toxin B treatment of HEK cells potently and efficiently blocked mAChR-stimulated PLD. In contrast, basal and phorbol ester-stimulated PLD activities were not or only slightly reduced. Cytochalasin B and Clostridium botulinum C2 toxin, mimicking the effect of toxin B on the actin cytoskeleton but without involving Rho proteins, had no effect on mAChR-stimulated PLD. Toxin B did not alter cell surface mAChR number and mAChR-stimulated binding of (guanosine 5'-O-(thio)triphosphate (GTP gamma S) to G proteins. In addition to mAChR-stimulated PLD, toxin B treatment also inhibited PLD activation by the direct G protein activators, AlF4- and GTP gamma S, studied in intact and permeabilized cells, respectively. Finally, C. botulinum C3 exoenzyme, which ADP-ribosylates Rho proteins, mimicked the inhibitory effect of toxin B on GTP gamma S-stimulated PLD activity. In conclusion, the data presented indicate that toxin B potently and selectively interferes with receptor coupling mechanisms to PLD, and furthermore suggest an essential role for Rho proteins in receptor signaling to PLD.