Eukaryotic DNA topoisomcrase II is a ubiquitous nuclear enzyme essential for maintaining the correct conformation of DNA. The enzyme acts to catalyse changes in the tertian structure of DNA. via the introduction of transient double-stranded breaks. Mammalian cells express two isoforms of type II topoisomerase. designated topoisomerase IIα and topoisomerase IIβ, which display differential expression and intracellular localisation. Levels of topoisomerase IIα gene expression are elevated in rapidly proliferating cells, whereas the β isoform is expressed at approximately equal levels throughout the cell cycle. Protein products of the two isoforms of topoisomerase II found in human cells are the primary intracellular targets of many common, effective chemotherapy drugs. The development of drug resistance, however, is a major clinical problem caused by both enzymes. The levels of topoisomerase IIα and topoisomerase IIβ are important determinants for the sensitivity of cells to the cytoxicity of drugs, with down-regulation of topoisomerase II thought to be a major factor involved in drug resistance. The rate of transcription is the main mechanism for controlling topoisomerase II expression and activity, and this is achieved by the binding of transcription factors to specific regulatory elements within the promoter sequence. Molecular mechanisms responsible for the regulation of expression of the topoisomerase II enzymes are thought to be associated with resistance to chemotherapy drugs, and therefore understanding these mechanisms is an important research focus. This study reports the cloning and characterisation of a 1.5 kb fragment of the 5'- flanking and untranslated region of the topoisomerase IIβ promoter (-1357 to +122). Analyses of 5'-serially and internally deleted lueiferase reporter constructs revealed a region upstream of the transcription start site (-1357 to -1228). which could have a negative regulatory role, and suggested 55% of topoisomerase IIβ promoter activity could be attributed to the region between -654 and -456. Mutational analysis of putative regulatory elements indicated that the two inverted CCAAT box (ICB1 and ICB2) within the latter region were important for regulation of topoisomerase IIβ promoter activity. Gel mobility shift assays indicated that both inverted CCAAT boxes in the promoter bound the transcription factor NF-Y. while ICB2 and a GC element were capable of binding transcription factors Sp1 and Sp3.