The role of topoisomerase II (topo II) in chromosome condensation was studied in a mitotic extract derived from Xenopus eggs by specific immunodepletion. HeLa nuclei, which have a high complement of endogenous topo II, are converted to mitotic chromosomes in the topo II-depleted extract equally well as in the control. Chicken erythrocyte nuclei, however, which have a very low content of topo II, do not convert to condensed chromosomes in the depleted extract, although their condensation is normal upon addition of purified topo II. Dosage experiments support the possible notion of a structural involvement of topo II in chromosome condensation. In the topo II-depleted extract the erythrocyte nuclei progress to precondensation chromosomes, which lack the nuclear membrane-lamina complex and consist of a cluster of swollen chromatids.