It is well known that certain bacterial toxins, e.g. cholera and pertussis toxins, ADP-ribosylate eukaryotic regulatory proteins. They have become invaluable tools in the study of G protein-linked receptors. Less well appreciated is the fact that certain strains of Clostridium botulinum types C and D produce an ADP-ribosyltransferase, termed C3. This enzyme is structurally and functionally distinct from botulinum neurotoxins C1 and D. Its substrate is the 21 kDa GTP-binding protein rho. Klaus Aktories and Alan Hall explain why C3 is now an important tool in analysing the regulatory function of the ras-related protein rho.