Evidence is presented that the nitroxide free radical, TEMPO, at concentrations commonly used to prevent oxidative damage, increases the intracellular hydrogen peroxide concentration. To investigate the origin of this increased hydrogen peroxide concentration, we have incubated various human tumor cell lines with compounds interfering with the generation of active oxygen metabolites. Sodium azide, inhibitor of the respiratory chain, the iron-chelating agent desferrioxamine, superoxide dismutase and catalase had no effect on the hydrogen peroxide concentration. Metyrapone, inhibitor of the cytochrome P450 system, was demonstrated to decrease, but not completely prevent, the hydrogen peroxide production. N-ethylmaleimide, a sulphydryl-bond alkylating agent, was able to completely prevent the increased hydrogen peroxide production. We conclude that, by increasing the cellular hydrogen peroxide concentration, TEMPO exerts a pro-oxidant effect. This increase in hydrogen peroxide production seems to be mediated by the induction of oxidase activity in the cytochrome P450 system, but other cellular systems involved in electron transport may also play a role.