TopoisomeraseII (Topo II) is a major component of chromosomal scaffolds and essential for mitotic chromosome condensation, but the mechanism of this action remains unknown. Here, we used an in vitro chromatin reconstitution system in combination with atomic force and fluorescence microscopic analyses to determine how Topo II affects chromosomal structure. Topo II bound to bare DNA and clamped the two DNA strands together, even in the absence of ATP. In addition, Topo II promoted chromatin compaction in a manner dependent on histone H1 but independent of ATP. Histone H1-induced 30-nm chromatin fibers were converted into a large complex by Topo II. Fluorescence microscopic analysis of the Brownian motion of chromatin stained with 4',6-diamidino-2-phenylindole showed that the reconstituted chromatin became larger following the addition of Topo II in the presence but not the absence of histone H1. Based on these findings, we propose that chromatin packing is triggered by histone H1-dependent, Topo II-mediated clamping of DNA strands.