Purified Drosophila indirect-flight-muscle actin and arthrin, an actin-ubiquitin conjugate, were ADP-ribosylated by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin. Phalloidin treatment inhibited the ADP-ribosylation of Drosophila actin and arthrin. Like actin, the ADP-ribose-arthrin linkage was sensitive towards hydroxylamine treatment, indicating arginine as the amino acid acceptor. Actin translated in vitro from the indirect-flight-muscle-specific gene Act88F was ADP-ribosylated by C. botulinum C2 toxin and C. perfringens iota toxin. Actin from the R177Q mutant of Act88F translated in vivo was not ADP-ribosylated confirming Arg-177 as the ADP-ribose acceptor. Mutant L176M actin was modified by both toxins, indicating that amino acid 176 of actin does not define the substrate specificity of C. botulinum C2 toxin. Whereas the gene products of various C-terminal mutants of Act88F translated in vitro (E334K, V339I, E364K, G368E, R372H) were substrates for ADP-ribosylation by C. botulinum C2 toxin and by C. perfringens iota toxin, neither toxin modified the N-terminal O-12 deletion mutant.