Isolated cytoplasmic actin of human platelet and pig liver actin, but not rabbit skeletal muscle actin, was ADP-ribosylated by botulinum C2 toxin in the presence of [32P]NAD. Tryptic digestion of the [32P]ADP-ribosylated platelet actin generated two labeled peptides: a soluble peptide covering residues 174-183 and an insoluble fragment containing residues 148-183. Further digestion of these two peptides with thermolysin yielded the same radioactive peptide, which was in both cases peptide 175-177. Amino acid sequence analysis of peptides 174-183 and 175-177 located the ADP-ribosylation on Arg177.