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The antitumour activity of actinomycin D (AMD) has been proposed to result, in part, from its intercalation into DNA dG-dC base-pairs leading to an inhibition of RNA synthesis. We have recently prepared 2-deamino-2-nitroactinomycin D and 2-deamino-actinomycin D and determined that, unlike AMD, these analogues do not intercalate into calf-thymus DNA. In the present study we show that these analogues and their corresponding peptide-free diethylamino derivatives are more effective than the parent AMD in forming ion radicals, in stimulating oxygen uptake and in forming superoxide anion when incubated in the presence of NADPH and NADPH cytochrome P-450 reductase. NaBH4-mediated reduction of these compounds yielded free radicals as shown by electron paramagnetic resonance (e.p.r.) spectroscopy. Free radicals could also be generated by incubation of these actinomycins with NADPH and either liver microsomes or purified NADPH cytochrome P-450 reductase. In the presence of molecular oxygen these free radicals spontaneously reoxidized by transfer of a single electron to molecular oxygen to form superoxide. Relative rates of superoxide formation were established for these substrates with the 2-deamino-2-nitroactinomycin D exhibiting the highest activity. It is proposed that the antitumour activity of these AMD analogues results, in part, from their ability to form reactive reduced oxygen species and, as such, these actinomycin derivatives may serve as useful probes for the tumouricidal mechanism of this family of agents.