RhoA, -B, and -C are ADP-ribosylated and biologically inactivated by Clostridium botulinum C3 exoenzyme and related C3-like transferases. We report that RalA GTPase, which is not ADP-ribosylated by C3, inhibits ADP-ribosylation of RhoA by C3 from C. botulinum (C3bot), Clostridium limosum (C3lim), and Bacillus cereus (C3cer) but not from Staphylococcus aureus (C3stau) in human platelet membranes and rat brain lysate. Inhibition by RalA occurs with the GDP- and guanosine 5'-3-O-(thio)triphosphate-bound forms of RalA and is overcome by increasing concentrations of C3. A direct interaction of RalA with C3 was verified by precipitation of the transferase with GST-RalA-Sepharose. The affinity constant (K(d)) of the binding of RalA to C3lim was 12 nm as determined by fluorescence titration. RalA increased the NAD glycohydrolase activity of C3bot by about 5-fold. Although RalA had no effect on glucosylation of Rho GTPases by Clostridium difficile toxin B, C3bot and C3lim inhibited glucosylation of RalA by Clostridium sordellii lethal toxin. Furthermore, C3bot decreased activation of phospholipase D by RalA. The data indicate that several C3 exoenzymes directly interact with RalA without ADP-ribosylating the GTPase. The interaction is of high affinity and interferes with essential functions of C3 and RalA.