The effects of theonellamide F, a marine bicyclic peptide, on vacuolar formation in cultured cells were studied. Theonellamide F induced large vacuoles in 6 types of mammalian cells. The vacuoles induced by theonellamide F in 3Y1 cells accumulated acridine orange, a fluorescent probe indicating the presence of an acidic organelle. Their disappearance following treatment with bafilomycin A1 suggests that these vacuoles contain vacuolar ATPase to maintain an acidic internal milieu, and this is similar to those induced by Helicobacter pylori toxin VacA. The vacuoles induced by theonellamide F were not significantly decreased in size or number by nocodazole treatment, and the localization of a small GTPase, rab7, did not always correspond to the outline of the vacuoles. These results suggest that the molecular mode of action of vacuolar formation by theonellamides may differ from that by VacA and can be considered unique.