Various C3-like ADP-ribosyltransferases like Clostridium botulinum exoenzyme C3, C limosum transferase, B cereus transferase and a transferase from Staphylococcus aureus (EDIN) selectively modify the low-molecular mass GTP-binding proteins RhoA,B,C. UV-irradiation of C limosum transferase in the presence of [carbonyl-14C]NAD resulted in radiolabeling of Glu-174. Concomitantly, ADP-ribosyltransferase and NAD glycohydrolase activities were inhibited. Site-directed mutagenesis of Glu-174 (E174D, E174Q) which resulted in more than 1000-fold reduction of enzyme activity, suggests that the glutamic acid residue is essentially involved in the catalytic action of C3-like transferases. These findings support the view that all bacterial ADP-ribosyltransferases share a similar active-site structure.