The Clostridium botulinum C2 toxin ADP-ribosylates monomeric actin, thereby inducing disassembly of actin filaments, alteration of focal adhesions, and rounding of cells. After treatment with C2 toxin, cells stop to proliferate but remain viable for about 2 days. In view of reported correlations between the structure of the actin cytoskeleton and cell cycle transition, the effects of C2 toxin on the G(2)/M phase transition of the cell division cycle were studied. Since C2 toxin delayed entry into mitosis in HeLa cells, those enzymes which control entry into mitosis, the cyclin-dependent protein kinase mitosis-promoting factor (MPF) and the phosphatase cdc25-C were examined after treatment of synchronized cells with C2 toxin. MPF is composed of the regulatory cyclin B and the enzymatic p34cdc2 kinase subunits. For its activation at the G2/M border, p34cdc2 needs to be associated with cyclin B and additionally dephosphorylated at Tyr-15 by the specific phosphatase cdc25-C. Treatment of synchronized cells in S or G2 phase with C. botulinum C2 toxin prevented p34cdc2 protein kinase activation by inhibiting its tyrosine dephosphorylation at the G2/M border. Furthermore, the activity of cdc25-C phosphatase was decreased after treatment of cells with C2 toxin. Our results suggest that the prevented activation of the mitotic inducers p34cdc2 kinase and cdc25-C phosphatase represents the final downstream events in the action of C2 toxin resulting in a G(2) phase cell cycle delay in synchronized HeLa cells.