Antineoplastic bis(dioxopiperazine)s, such as meso-2,3-bis(2,6-dioxopiperazin-4-yl)butane (ICRF-193), are widely believed to be only catalytic inhibitors of topoisomerase II. However, topoisomerase inhibitors have little or no antineoplastic activity unless they are topoisomerase poisons, a special subclass of topoisomerase-targeting drugs that stabilize topoisomerase-DNA strand passing intermediates and thus cause the topoisomerase to become a cytotoxic DNA-damaging agent. Here we report that ICRF-193 is a very significant topoisomerase II poison. Detection of topoisomerase II poisoning by ICRF-193 required the use of a chaotropic protein denaturant in the topoisomerase poisoning assays. ICRF-193 caused dose-dependent cross-linking of human topoisomerase IIβ to DNA and stimulated topoisomerase IIβ-mediated DNA cleavage at specific sites on 32P-end-labeled DNA. Human topoisomerase IIα-mediated DNA cleavage was stimulated to a lesser extent by ICRF-193. In vivo experiments with MCF-7 cells also showed the requirement of a chaotropic protein denaturant in the assays and selectivity for the β-isozyme of human topoisomerase II. Studies with two topoisomerase IIβ-negative cell model systems confirmed significant topoisomerase II poisoning by ICRF-193 in the wild type cells and were consistent with β-isozyme selectivity. Common use of only the detergent, SDS, in assays may have led to failure to detect topoisomerase II poisoning by ICRF-193 in earlier studies.