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Cytochalasin D which shows marked cytotoxic effects on multi-tumor cells was newly isolated at high content(5.28 mg/g, dry weight)from Fungus Hypocrella bambusae(B.et Br.) Sacc. Its structure was elucidated by spectroscopic methods. Two-dimensional NMR techniques were applied to make complete assignment for the 1H- and 13C-NMR chemical shifts of this compound.