The efficacy of all chemotherapeutic agents is limited by the occurrence of drug resistance. To further understand resistance to topoisomerase (topo) II inhibitors, 50 sublines were isolated as single clones from parental cells by exposure to ETP or m-AMSA. Subsequently, a population of cells from each subline was exposed to three-fold higher drug concentrations allowing 16 stable sublines to be established at higher extracellular drug concentration. The frequency and nature of mutations in topo II in the drug selected cell lines have been evaluated. In order to screen a large number of cell lines, an RNase protection assay was developed. Fragments covering the entire coding sequence of topo II was isolated after PCR amplification and subcloned in pGEM3Z vector. Using this approach, mismatches was observed in 13.6% of resistant cell lines (12% of resistant cell lines exposed to lower drug concentrations and 18.8% of resistant cell lines exposed to higher drug concentrations). Our findings suggest that mutations of topo II gene seem to be an important and frequent mechanism of resistance to topo II inhibitors.