Aliphatic amine N-oxides have long been identified as non-toxic metabolites of a large number of tertiary amines drugs. Bioreduction of such N-oxides will generate the active parent amine. This principle has been adopted to develop AQ4N, a di-N-oxide anticancer prodrug with little intrinsic cytotoxicity. However, AQ4N is bioreduced in hypoxic regions of solid tumors and micrometastatic deposits to generate a cytotoxic alkylaminoanthraquinone metabolite. The 4-electron reduction metabolite of AQ4N has high affinity for DNA and is a potent inhibitor of topoisomerase II, a DNA processing enzyme crucial to cell division. The development of AQ4N has proceeded on many fronts in order to establish this unique anticancer prodrug opportunity. Preclinical studies in vivo have demonstrated that although AQ4N has little or no intrinsic cytotoxic activity per se it (i) enhances the antitumor effects of radiation and conventional chemotherapeutic agents, (ii) is pharmacokinetically stable, and (iii) is a substrate for cytochrome P450 (CYP). A study of AQ4N metabolism in vitro and ex vivo using purified CYP enzymes, phenotyped human livers and CYP transfected cell lines shows that CYP3A, 1A and 1B1 family members contribute to AQ4N bioreduction in the absence of oxygen. Importantly AQ4N is shown to be metabolized by tumors known to express CYP isoforms. AQ4N is currently in Phase I clinical trials.