Clostridium botulinum exoenzyme C3 exclusively ADP-ribosylates RhoA, B, and C to inactivate them, resulting in disaggregation of the actin filaments in intact cells. The ADP-ribose resides at Asn-41 in the effector binding region, leading to the notion that ADP-ribosylation inactivates Rho by blocking coupling of Rho to its downstream effectors. In a recombinant system, however, ADP-ribosylated Rho bound to effector proteins such as phospholipase D-1 (PLD1), Rho-kinase (ROK), and rhotekin. The ADP-ribose rather mediated binding of Rho-GDP to PLD1. ADP-ribosylation of Rho-GDP followed by GTP-gamma-S loading resulted in binding but not in PLD activation. On the other hand, ADP-ribosylation of Rho previously activated by binding to GTP-gamma-S resulted in full PLD activation. This finding indicates that ADP-ribosylation seems to prevent GTP-induced change to the active conformation of switch I, the prerequisite of Rho-PLD interaction. In contrast to recombinant systems, ADP-ribosylation in intact cells results in functional inactivation of Rho, indicating other mechanisms of inactivation than blocking effector coupling.