Clostridium histolyticum vacuolating cytotoxin was partially purified from culture broth using ammonium sulfate precipitation, gel filtration and hydrophobic interaction chromatography. The toxin caused vacuolization of HeLa cells visible under a light microscope after 2 h and distinct after 8 h. Transmission electron microscopy revealed the presence of numerous vacuoles, condensation of the mitochondrial matrix, increased cytoplasm density and increased amounts of heterochromatin. Apoptosis was not detected either by electron microscopy or by an apoptosis/necrosis discrimination assay with fluorescein-labeled annexin V and propidium iodide, or DNA fragmentation assay. Calcium ion influx was detected by flow cytometry after labeling cells with Fluo-4 AM. Vacuolation of HeLa cells by C. histolyticum cytotoxin was inhibited by bafilomycin A1, suggesting involvement of H+ -ATPase in the formation of vacuoles.