A number of quinolones and related antibacterial compounds were screened for activity against calf thymus topoisomerase II by using the P4 unknotting and DNA breakage assays. Several compounds from different structural classes which inhibited DNA unknotting with 50% inhibitory concentrations ranging from 8 to 25 micrograms/ml were identified. Two experimental isothiazoloquinolones from this group, designated A-65281 and A-65282, were also found to induce considerable DNA breakage mediated by calf thymus topoisomerase II, with 32P-end-labeled pBR322 as the substrate. These compounds were nearly as potent as teniposide, with DNA breakage activity evident at concentrations as low as 4 micrograms/ml. However, some differences in DNA cleavage patterns from those with teniposide were evident. These studies have thus identified a new class of agents which have activity against both bacterial and eukaryotic type II topoisomerases. The implications of these data for the selectivity of topoisomerase-directed compounds and the potential toxicity of such compounds developed as antibacterial agents are discussed.