Topoisomerase II (Topo II) that decatenates newly synthesized DNA is targeted by many anticancer drugs. Some of these drugs stabilize intermediate complexes of DNA with Topo II and others act as catalytic inhibitors of Topo II. Simultaneous depletion of Topo IIα and Topo IIβ, the two isoforms of mammalian Topo II, prevents cell growth and normal mitosis, but the role of Topo II in other phases of mammalian cell cycle has not yet been elucidated. We have developed a derivative of p53-suppressed human cells with constitutive depletion of Topo IIβ and doxycycline-regulated conditional depletion of Topo IIα. The effects of Topo II depletion on cell cycle progression were analyzed by time-lapse video microscopy, pulse-chase flow cytometry and mitotic morphology. Topo II depletion increased the duration of the cell cycle and mitosis, interfered with chromosome condensation and sister chromatid segregation and led to frequent failure of cell division, ending in either cell death or restitution of polyploid cells. Topo II depletion did not change the rate of DNA replication but increased the duration of G 2. These results define the effects of decreased Topo II activity, rather than intermediate complex stabilization, on the mammalian cell cycle.