The lethal and edema-inducing alpha-toxin from Clostridium novyi causes rounding up of cultured cell lines by redistribution of the actin cytoskeleton. alpha-Toxin belongs to the family of large clostridial cytotoxins that encompasses Clostridium difficile toxin A and B and the lethal toxin from Clostridium sordellii. Toxin A and toxin B have been recently identified as monoglucosyltransferases to modify the low molecular mass GTPases of the Rho subfamily (Just, I., Selzer, J., Wilm, M., Von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) Nature 375, 500-503 and Just, I., Wilm, M., Selzer, J., Rex, G., Von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) J. Biol. Chem. 270, 13932-13936). We report here the identification of the alpha-toxin-catalyzed modification of Rho. Using electrospray mass spectrometry, the mass of the modification was determined as 203 Da, consistent with a N-acetyl-hexosamine moiety. UDP-N-acetyl-glucosamine selectively served as cosubstrate for alpha-toxin-catalyzed modification into the Rho subfamily proteins Rho, Rac, Cdc42, and RhoG. The acceptor amino acid of N-acetyl-glucosaminylation was identified by mutagenesis as Thr-37 in Rho (equivalent to Thr-35 in Rac/Cdc42), which is located in the effector domain of the GTPases. C. novyi alpha-toxin seems to mediate its cytotoxic effects on cells by mimicking endogenous post-translational modification of cellular proteins.