Oxolinic acid forms complexes with gyrase and DNA in such a way that subsequent denaturation of gyrase reveals DNA cleavage. Cleavage sites were mapped in a 10,000 base-pair region of the Escherichia coli chromosome containing the dnaA, dnaN, recF, and gyrB genes. Twenty-four cleavage sites were identified. The sites were cleaved at different frequencies, with the most frequent cleavage occurring within gyrB. Not all sites were equally sensitive to oxolinic acid concentration, some sites exhibited an altered cleavage frequency when the gyrB225 delta topA mutant strain DM800 was compared with wild-type cells, and coumermycin selectively changed the cleavage frequency at a few sites in the mutant strain DM800. These perturbations appear to alter the frequency of cleavage at a site but not the location of the site. The availability of many sites of differing strengths may be an important factor in the ability of gyrase to fine-tune the level of supercoiling or provide local swivels in bacterial DNA.