We have constructed a plasmid which contains 22 copies of a 147 bp DNA fragment which contains the major DNA gyrase cleavage site from plasmid pBR322 (located at base-pair 990). We have found that this fragment is efficiently bound and cleaved by gyrase. The selectivity for the sequence corresponding to position 990 in pBR322 is maintained even when this site is located only 15 bp from one end of the 147 bp fragment. A strategy for the specific incorporation of a single thiophosphoryl linkage into the 147 bp fragment has been developed, and gyrase has been shown to catalyse efficient cleavage of fragments bearing phosphorothioate linkages at the gyrase cleavage site in one or both strands.