The formation of a topoisomerase-quinolone-DNA ternary complex leads to cell death. We show here that an active strand breakage and reunion activity is required for formation of a norfloxacin-topoisomerase IV-DNA ternary complex that can arrest the progression of replication forks in vitro. Mutant topoisomerases containing either an active site mutation, a quinolone resistance-conferring mutation, or both, could all bind DNA as well as the wild-type, but unlike the wild-type, could not halt replication fork progression. The collision between the replication fork and the frozen topoisomerase converted the cleavable complex to a nonreversible form but did not generate a double-stranded break. Thus, the cytotoxicity of this class of topoisomerase inhibitors likely results from a two-step process: (i) conversion of the frozen topoisomerase-quinolone-DNA ternary complex to an unreversible form; and (ii) generation of a double-strand break by subsequent denaturation of the topoisomerase, perhaps by an aborted repair attempt.