Botulinum C2 toxin ADP-ribosylates actin in [32P]orthophosphate-labelled intact chick embryo cells (CEC). The toxin-induced rounding up of CEC is correlated with ADP-ribosylation of actin in intact cells in a time and concentration-dependent manner. Both, rounding up of cells and actin ADP-ribosylation, depend on the presence of both components of botulinum C2 toxin (components I and II) and are independent of the ability of CEC to divide. Treatment of CEC with botulinum C2 toxin induced a time-dependent disorganization of the typical architecture of the microfilament network as shown by fluorescein-phalloidin staining. Botulinum C2 toxin decreased the amount of Triton X-100 insoluble actin, while the fraction of Triton soluble actin was increased. Actin, which was 32P-labelled by botulinum C2 toxin in intact CEC, was recovered in the Triton soluble but not in the Triton insoluble actin fraction. It is suggested that in intact CEC botulinum C2 toxin causes ADP-ribosylation of G-actin but not of F-actin thereby leading to an accumulation in the pool of monomeric actin.