A novel ADP‐ribosyltransferase C3 was purified to homogeneity from filtrates of certain strains of Clostridium botulinum type C by ammonium sulfate precipitation, gel filtration, ion‐exchange chromatography and heat treatment. The molecular mass of botulinum ADP‐ribosyltransferase C3 was found to be 25 kDa. In the presence of [³²P]NAD but not with [carbonyl‐¹⁴C]NAD, C3 labelled 21–24‐kDa protein(s) in membranes of human platelets and other tissues. The K_m value of the ADP‐ribosylation reaction for NAD was about 2 μM. Labelling of the 21–24‐kDa protein(s) by C3 was largely reduced by addition of nicotinamide. Snake venom phosphodiesterase cleaved the ADP‐ribose attached to the 21–24‐kDa protein(s) by C3 and released 5'AMP. C3 catalyzed hydrolysis of [carbonyl‐¹⁴C]NAD and released [carbonyl‐¹⁴C]nicotinamide. ADP‐ribosylation of 21–24‐kDa platelet membrane protein(s) was biphasically regulated by Mg²⁺, Mn²⁺ and Ca²⁺. In the absence of free divalent cations GTP, GTP [γS] and GDP but not GDP[βS], GMP, ATP or ATP[γS] increased labelling by C3. In the presence of Mg²⁺, GTP[γS] was inhibitory. Guanine nucleotides prevented heat inactivation of the substrate protein(s) with the rank order GTP[γS] = GTP = GDP > GDP[βSL] > GMP ≫ ATP = ATP[γS]. The data support the view that the novel ADP‐ribosyltransferase C3 modifies eukaryotic 21–24‐kDa GTP‐binding protein(s).