The decatenation activity of DNA topoisomerase II is essential for viability as 
eukaryotic cells traverse mitosis. Phosphorylation has been shown to stimulate 
topoisomerase II activity in vitro. Here we show that topoisomerase II is a 
phosphoprotein in yeast and that the level of incorporated phosphate is 
significantly higher at mitosis than in G1. Comparison of tryptic phosphopeptide 
maps reveals that the major phosphorylation sites in vivo are targets for casein 
kinase II. Incorporation of phosphate into topoisomerase II is nearly 
undetectable at the non-permissive temperature in a conditional casein kinase II 
mutant. The sites modified by casein kinase II are located in the extreme 
C-terminal domain of topoisomerase II. This domain is absent in prokaryotic and 
highly divergent among eukaryotic type II topoisomerases, and may serve to 
regulate functions of topoisomerase II that are unique to eukaryotic cells.