Using topoisomerase II (topo II) isozyme-specific antibodies, we investigated the phosphorylation of topo $II{alpha}$ in mitotic HeLa cells. Topo $II{alpha}$ was specifically modified in the mitotic cells, resulting in slow migration on SDS-polyacrylamide gel electrophoresis. To characterize the nature of this modification, we treated the nuclear extracts prepared from the mitotic cells with alkaline phosphatase. After the treatment with alkaline phosphatase, the slowly migrated band disappeared and instead a normal (170 kDa) topo $II{alpha}$ band appeared. These results indicate that human topo $II{alpha}$ is modified at a specific site(s) in M phase by phosphorylation, supporting the possibility that M phase-specific phosphorylation of topo II is critical for mitotic chromosome condensation and segregation.