Using topoisomerase (I (topo II) isozyme-specific antibodies, we investigated the phosphocylation of topo Ila in mitotic HeLa cells. Topo IIa 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 IIa band appeared. These results indicate that human topo IIa is modified at a specific sites) in M phase by phosphorylation, supporting the possibility that M phase-specific phosphorylation of topo II is critical for mitotic chromosome condensation and segregation.