When bound to DNA, topoisomerase II from Saccharomycescerevisiae exhibits burst kineticswith respect to ATP hydrolysis. Pre-steady-state analysis showsthat the enzyme binds and hydrolyzestwo ATP per reaction cycle. Our data indicate that at least one ofthe two ATP is rapidly hydrolyzedprior to the rate-determining step in the reaction mechanism. WhenDNA is not bound to topoisomeraseII, the rate-determining step shifts to become either ATP binding orhydrolysis. Two possible mechanismsare proposed that agree with our observations.