We have examined the long-range distribution of double-stranded DNA cleavage sites for Drosophila melanogaster topoisomerase II. These studies reveal a novel partitioning of preferred topoisomerase II cleavage sites. In the eukaryotic DNAs examined, major cleavage sites were typically found in nontranscribed spacer segments and close to the 5' and 3' boundaries of genes. In contrast, there were few if any prominent cleavage sites within genes. In addition, most of the major topoisomerase II cleavage sites closely corresponded to naked DNA hypersensitive sites for the prokaryotic enzyme, micrococcal nuclease.