DNA topoisomerase II copurifies with and is phosphorylated by protein kinase CKII. In this study, a yeast two-hybrid system was used to investigate the interaction between human topoisomerase II isozymes and CKII subunits. The two-hybrid test clearly showed that both topoisomerase IIalpha and IIbeta interact with the CKIIbeta, but not the CKIIalpha subunit. The two-hybrid test also demonstrated that topoisomerase IIbeta residues 1099-1263 and topoisomerase IIalpha residues 1078-1182 mediate the interaction with the CKIIbeta subunit, providing evidence that the leucine zipper motif and the major CKII-dependent phosphorylation sites of topoisomerase II are unnecessary for its physical binding to CKIIbeta. Furthermore, a DNA relaxation assay demonstrated that the CKII subunit enhances topoisomerase II activity by physical interaction with topoisomerase II.