C3 exoenzymes (members of the ADP-ribosyltranferase family) are produced by Clostridium botulinum (C3bot1 and -2), Clostridium limosum (C3lim), Bacillus cereus (C3cer), and Staphylococcus aureus (C3stau1-3). These exoenzymes lack a translocation domain but are known to specifically inactivate Rho GTPases in host target cells. Here, we report the crystal structure of C3bot1 in complex with RalA (a GTPase of the Ras subfamily) and GDP at a resolution of 2.66 Å. RalA is not ADP-ribosylated by C3 exoenzymes but inhibits ADP-ribosylation of RhoA by C3bot1, C3lim, and C3cer to different extents. The structure provides an insight into the molecular interactions between C3bot1 and RalA involving the catalytic ADP-ribosylating turn-turn (ARTT) loop from C3bot1 and helix ␣4 and strand 6 (which are not part of the GDP-binding pocket) from RalA. The structure also suggests a molecular explanation for the different levels of C3-exoenzyme inhibition by RalA and why RhoA does not bind C3bot1 in this manner.ADP-ribosylation ͉ protein-protein interaction ͉ x-ray crystallography B acteria produce many enzymes that show extraordinary specificity for mammalian intracellular proteins. The specificity of these bacterial enzymes has not only made them a valuable tool for elucidating the cellular functions of their targets but has also increased our understanding of protein interactions. Clostridium botulinum is no exception, producing two classes of enzymes that have very specific protein targets, the neurotoxins A-G and the ADP-ribosyltransferases C2, C3bot1, and C3bot2. C2 and C3bot are part of a larger family of ADP-ribosylating toxins (1, 2), including diphtheria toxin and cholera toxin, which cleave NAD and transfer ADP-ribose to target proteins. Although the members of this family have homologous enzymatic domains and similar active sites, these toxins ADP-ribosylate and, therefore, disable a range of cellular targets.