The major type I DNA topoisomerase (topo I) has been purified from HeLa cell nuclei to a homogeneous, monomeric protein (Mr = 100,000). Similar to the nicking-closing enzyme (Mr = 67,000) from rat liver (Been, M. D., and Champoux, J. J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 2883-2887), HeLa topo I has the following properties: (a) HeLa topo I breaks down single-stranded DNA to smaller fragments, each with an enzyme-linked 3'-phosphoryl end and a free 5'-OH end. This cleavage is not dependent upon protein denaturant or protease treatment. (b) HeLa topo I produces single-stranded DNA circles from linear single-stranded DNA. Such DNA circles are believed to be produced by the intramolecular cyclization of topo I-linked, single-stranded DNA fragments. (c) HeLa topo I-linked, single-stranded fragments (donors) can join covalently to double-stranded DNA possessing a 5'-OH group (acceptors). The donor is transferred to the 5'-OH end of the acceptor, independent of the position of the end (internal nick or end of linear DNA) or the configuration of the end (flush, 5'-protruding, or 5'-recessed end) of the acceptor. (d) HeLa topo I cleavage of single-stranded DNA is site-specific, but no special sequence at the ends of the acceptor molecule is apparently required for a successful heterologous strand transfer. These results suggest that HeLa topo I may be involved in DNA sequence rearrangements in addition to its possible role as a swivelase for transcription and replication.