DNA topoisomerase (topo) I is a nuclear enzyme that plays an important role in DNA metabolism. Based on conserved nuclear targeting sequences, four classic nuclear localization signals (NLSs) have been proposed at the N terminus of human topo I, but studies with yeast have suggested that only one of them (amino acids (aa) 150 -156) is sufficient to direct the enzyme to the nucleus. In this study, we expressed human topo I fused to enhanced green fluorescent protein (EGFP) in mammalian cells and demonstrated that whereas aa 150 -156 are sufficient for nuclear localization, the nucleolar localization requires aa 157-199. More importantly, we identified a novel NLS within aa 117-146. In contrast to the classic NLSs that are rich in basic amino acids, the novel NLS identified in this study is rich in acidic amino acids. Furthermore, this novel NLS alone is sufficient to direct not only EGFP into the nucleus but also topo I; and the EGFP⅐topo I fusion driven by the novel NLS is as active in vivo as the wild-type topo I in response to the topo I inhibitor topotecan. Together, our results suggest that human topo I carries two independent NLSs that have opposite amino acid compositions.