CP-115,953 [6,8-difluoro-7-(4'-hydroxyphenyl)-1-cyclopropyl-4- quinolone-3-carboxylic acid] is a novel quinolone that is highly active against topoisomerase II in vitro and in mammalian cells in culture (M. J. Robinson, B. A. Martin, T. D. Gootz, P. R. McGuirk, M. Moynihan, J. A. Sutcliffe, and N. Osheroff, J. Biol. Chem. 266:14585-14592, 1991). However, the features of the drug that contribute to its activity towards mammalian systems have not been characterized. Therefore, CP-115,953 and a series of related quinolones were examined for their activity against calf thymus topoisomerase II and cultured mammalian cells. CP-115,953 stimulated DNA cleavage mediated by the type II enzyme with a potency that was approximately 600-fold greater than that of the antimicrobial quinolone ciprofloxacin and approximately 50-fold greater than that of the antineoplastic drug etoposide. As determined by the ability to enhance enzyme-mediated DNA cleavage, quinolone activity towards calf thymus topoisomerase II was enhanced by the presence of a cyclopropyl group at the N-1 ring position and by the presence of a fluorine at C-8. Furthermore, the 4'-hydroxyphenyl substituent at the C-7 position was critical for the potency of CP-115,953 towards the mammalian type II enzyme. In this regard, the aromatic nature of the C-7 ring as well as the presence and the position of the 4'-hydroxyl group contributed greatly to drug activity. Finally, the cytotoxicity of quinolones in the CP-115,953 series towards mammalian cells paralleled the in vitro stimulation of DNA cleavage by topoisomerase II rather than the inhibition of enzyme-catalyzed DNA relaxation. This correlation strongly suggests that these quinolones promote cell death by converting topoisomerase II to a cellular poison.