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5H-Pyridophenoxazin-5-one (PPH), a new anticancer iminoquinone, is able to inhibit a large number of lymphoblastoid and solid tumor-derived cells at submicromolar concentrations. Molecular modeling calculations indicated that this compound might intercalate into the DNA double strand. This was also supported by nuclear magnetic resonance studies. Since free radicals arising from anticancer quinonic drugs have been proposed to be key species responsible for DNA cleavage, we have aimed to intercept and identify free radicals from PPH generated under bioreductive conditions. The first and second monoelectronic reduction potentials of PPH were measured by means of cyclic voltammetry: the reduction potential of PPH is compatible with its reduction by compounds such as NADH, and suggested that reduction of PPH may play a role in its cytotoxicity. The radical anion PPH(*)(-) was detected by means of electron paramagnetic resonance spectroscopy, and its identification was supported by DFT calculations. EPR experiments in the presence of spin traps 5,5-dimethylpyrroline N-oxide and 5-(diethoxyphosphoryl)-5-methylpyrroline N-oxide suggested the occurrence of an electron transfer between the radical anion of the drug and oxygen resulting in the formation of the superoxide anion (O(2)(*)(-)). The enthalpy of the reaction of PPH(*)(-) with O(2) was determined both in the gas phase and in solution at the B3LYP/6-31+G level using the isodensity PCM method, and the overall process in dimethyl sulfoxide was predicted to be slightly exothermic. We propose that the monoelectronic reduction of PPH in the proximity of DNA may eventually lead to radicals that could cause considerable damage to DNA, thus accounting for the high cytotoxic activity of the drug. Indeed, a comet assay (alkaline single-cell electrophoresis) showed that PPH causes free radical-induced DNA damage.