Type II topoisomerases catalyze decatenation of the catenated network of kinetoplast DNA [J. C. Marini, K. G. Miller, and P. T. Englund (1980) J. Biol. Chem. 255, 4976-4979]. The individual DNA circles and small catenanes produced during the decatenation reaction can be separated from the large network of substrate DNA by 5 min centrifugation at 13,000g and quantitated. The appearance of these decatenated DNA molecules which appear in the supernatant first showed a lag, whose duration depended on the enzyme concentration, and then increased linearly with time until it reached a plateau. The slope of the linear part of the kinetic curve was directly proportional to the enzyme concentration, whether a purified or crude preparation of type II topoisomerase from mammalian cells was used. These findings led us to a rapid quantitative assay of type II topoisomerases not involving electrophoresis. The method was developed with purified enzyme but was also useful for assay of the activity in crude extracts. Surprisingly, the type I topoisomerase, even when present in large excess, failed to decatenate the nicked DNA circles often present in the kinetoplast DNA. This renders the assay virtually free from interference by type I enzyme. The method is sensitive and allowed quantitative estimation of the enzyme activity present in the crude extracts corresponding to that derived from 500 to 700 cultured mammalian cells. Since various type II topoisomerases from procaryotic, eucaryotic, and viral sources decatenate kinetoplast DNA and generate similar DNA products, the assay method is likely to be generally applicable.