Two isoforms, 1 and 2, of human DNA topoisomerase III beta were expressed in HeLa cells as a fusion protein to the C-terminus of green fluorescent protein (GFP). The fusion protein of the isoform 1 was found to be localized to the nucleus, and to be associated with chromosomes during metaphase and anaphase. As yeast top3 mutants are known to exhibit phenotypes indicative of defective chromosome segregation, the result suggests that the isoform I of the human enzyme may also be involved in chromosome segregation. Two-hybrid screening for interaction partners of the isoform identified three candidate genes: CENP-F, a gene encoding a centromere protein and two genes of no known function, one of which was novel. The GFP fusion of the isoform 2 was found in the cytoplasm, indicating the nuclear localization signal sequence in the isoform I is in the C-terminal part that is different between the two isoforms.