The cytotoxicity and effect of NB901 (one of mono-nucleoside derivative)on pBR322 DNA cleavage reaction by topoisotnerase Ⅱ werestudied. The results showed that 50% inhibiting concentration (IC50)of the drug against human hepatoma cell Bel 7402 and lung cancer cell PGT by neutral red colorimetry test in vitro were 7. 32 and 3. 53 μmol/L respectively. The presence of NB901 (5—10μmol/L)markedly stimulated DNA cleavage by 5-fold in the presence of 0. 2 U topoisornerase Ⅱ. It is suggested that topoisomerase Ⅱ be a possible target for NB901 which result in cytotoxicity.