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 Km value of the ADP-ribosylation reaction for NAD was about 2 microM. 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[gamma S] and GDP but not GDP[beta S], GMP, ATP or ATP[gamma S] increased labelling by C3. In the presence of Mg2+, GTP[gamma S] was inhibitory. Guanine nucleotides prevented heat inactivation of the substrate protein(s) with the rank order GTP[gamma S] = GTP = GDP greater than GDP[beta S] greater than GMP much greater than ATP = GMP = ATP[gamma S]. The data support the view that the novel ADP-ribosyltransferase C3 modifies eukaryotic 21-24-kDa GTP-binding protein(s).