It is known that topoisomerase IIα is phosphorylated by several kinases. To elucidate the role of phosphorylation of topoisomerase IIα in the cell cycle, we have examined the cell cycle behavior of phosphorylated topoisomerase IIα in HeLa cells using antibodies against several phospho-oligopeptides of this enzyme. Here we demonstrate that serine1212 in topoisomerase IIα is phosphorylated only in the mitotic phase. Using an antibody against an oligopeptide containing phosphoserine-1212 in topoisomerase IIα (PS1212), subcellular localization of topoisomerase IIα phosphorylated at serine1212 was examined by indirect immunofluorescence staining, and compared with that of overall topoisomerase IIα. Serine1212-phosphorylated topoisomerase IIα was localized specifically on mitotic chromosomes, but not on interphase chromosomes; this result contrasts with overall topoisomerase IIα which was observed on chomosomes in both interphase and mitosis. Serine1212-phosphorylated topoisomerase IIα first appeared on chromosome arms in prophase, became concentrated on the centromeres in metaphase, and disappeared in early telophase. In addition, ICRF-193, a catalytic inhibitor of topoisomerase II, prevented accumulation of serine1212-phosphorylated topoisomerase IIα at the centromeres. These results indicate that serine1212 of topoisomerase IIα is phosphorylated specifically during mitosis, and suggest that the serine1212-phosphorylated topoisomerase IIα acts on resolving topological constraint progressively from the chromosome arm to the centromere during metaphase chromosome condensation.