Benzene is a human carcinogen that induces hematopoietic malignancies. It is believed that benzene does not initiate leukemias directly, but rather generates DNA damage through a series of phenolic metabolites, especially 1,4-benzoquinone. The cellular consequences of 1,4-benzoquinone are consistent with those of topoisomerase II-targeted drugs. Therefore, it has been proposed that the compound initiates specific leukemias by acting as a topoisomerase II poison. This hypothesis, however, has not been supported by in vitro studies. While 1,4-benzoquinone has been shown to inhibit topoisomerase II catalysis, increases in enzyme-mediated DNA cleavage have not been reported. Because of the potential involvement of topoisomerase II in benzene-induced leukemias, we re-examined the effects of the compound on DNA cleavage mediated by human topoisomerase IIalpha. In contrast to previous reports, we found that 1,4-benzoquinone was a strong topoisomerase II poison and was more potent in vitro than the anticancer drug etoposide. DNA cleavage enhancement probably was unseen in previous studies due to the presence of reducing agents in reaction buffers and the incubation of 1,4-benzoquinone with the enzyme prior to the addition of DNA. 1,4-Benzoquinone increased topoisomerase II-mediated DNA cleavage primarily by enhancing the forward rate of scission. In vitro, the compound induced cleavage at DNA sites proximal to a defined leukemic chromosomal breakpoint and displayed a sequence specificity that differed from that of etoposide. Finally, 1,4-benzoquinone stimulated DNA cleavage by topoisomerase IIalpha in cultured human cells. The present findings are consistent with the hypothesis that topoisomerase IIalpha plays a role in the initiation of specific leukemias induced by benzene and its metabolites.