Co-application of the chemotherapeutic agents mitomycin C and doxorubicin has been shown to result in supra-additive tumor cell killing in vitro and in vivo. The interaction of mitomycin C and doxorubicin was found to be a true synergy, as defined by the Median Effect Analysis (MEA). In vitro evidence identified mitomycin C-derived formaldehyde and glutathione levels in the cell as potential players. Reactive oxygen species were not detected from the metabolism of mitomycin C, contrary to previous reports, probably due to the 10-fold lower drug concentration used herein. Since DNA cross-links were found to only increase additively with co-administration of the drugs, we propose that the poisoning of topoisomerase IIalpha by doxorubicin interacts with the drug-induced DNA cross-links. Therefore, the synergy observed between mitomycin C and doxorubicin may be elicited through mitomycin C-derived formaldehyde, glutathione depletion and the enhanced genotoxicity of the combination of DNA cross-links and topoisomerase IIalpha poisoning.