All eukaryotic forms of DNA topoisomerase I contain an extensive and highly charged N-terminal domain. This domain contains several nuclear localization sequences and is essential for in vivo function of the enzyme. However, so far no direct function of the N-terminal domain in the in vitro topoisomerase I reaction has been reported. In this study we have compared the in vitro activities of a truncated form of human topoisomerase I lacking amino acids 1-206 (p67) with the full-length enzyme (p91). Using these enzyme forms, we have identified for the first time a direct role of residues within the N-terminal domain in modulating topoisomerase I catalysis, as revealed by significant differences between p67 and p91 in DNA binding, cleavage, strand rotation, and ligation. A comparison with previously published studies showing no effect of deleting the first 174 or 190 amino acids of topoisomerase I (Stewart, L., Ireton, G. C., and Champoux, J. J. (1999) J. Biol. Chem. 274, 32950-32960; Bronstein, I. B., Wynne-Jones, A., Sukhanova, A., Fleury, F., Ianoul, A., Holden, J. A., Alix, A. J., Dodson, G. G., Jardillier, J. C., Nabiev, I., and Wilkinson, A. J. (1999) Anticancer Res. 19, 317-327) suggests a pivotal role of amino acids 191-206 in catalysis. Taken together the presented data indicate that at least part(s) of the N-terminal domain regulate(s) enzyme/DNA dynamics during relaxation most probably by controlling non-covalent DNA binding downstream of the cleavage site either directly or by coordinating DNA contacts by other parts of the enzyme.