13S condensin is a multisubunit protein complex essential for mitotic chromosome condensation in Xenopus egg extracts. Purified 135 condensin introduces positive supercoils into DNA in the presence of topoisomerase I and adenosine triphosphate in vitro. The supercoiling activity of 13S condensin was regulated by mitosis-specific phosphorylation. Immunodepletion, in vitro phospholylation, and peptide-mapping experiments indicated that Cdc2 is Likely to be the kinase that phosphorylates and activates 135 condensin. Multiple Cdc2 phosphorylation sites are clustered in the carboxyl-terminal domain of the XCAP-D2 (Xenopus chromosome-associated polypeptide D2) subunit. These results suggest that phosphorylation of 135 condensin by Cdc2 may trigger mitotic chromosome condensation in vitro.