Most cytochrome P450 (P450)-catalyzed reactions are believed to involve an FeO3+ intermediate as the actual oxygenating species. However, studies on the mechanism of steroid aromatization and subsequent model work have provided evidence that a peroxo-iron form (formally FeO2) can be involved directly in some oxidations. The possible involvement of peroxoiron was considered in P450-catalyzed N-oxygenations, because there is precedent for the use of H2O2 and organic peroxides in such reactions in the literature concerning synthetic and flavin reactions. The approach used was to compare P450 reactions involving the normal NADPH/NADPH-P450 reductase/O2 system with those supported by the oxygen surrogates H2O2 (which can directly form FeO2 and subsequently FeO3+) and iodosylbenzene (which can form FeO3 but not FeO2+). Iodosylbenzene was effective in supporting rabbit P450 1A2-catalyzed N,N-dimethyl-2-aminofluorene N-oxygenation, human P450 3A4-catalyzed quinidine N-oxygenation, rat P450 2B1-catalyzed oxidation of N-benzyl-(1-phenyl) cyclobutylamine to the N-hydroxyamine and nitrone, and rat P450 2B1-catalyzed and rabbit P450 2B4-catalyzed N-oxygenation of N,N-dimethylaniline (also N-demethylation). H2O2 also supported most of these reactions. A mutant of P450 2B4 with the substitution of alanine for threonine at position 302 has been shown to have decreased ability to catalyze reactions involving the putative FeO3+ but, presumably because of decreased ability to protonate the FeO2+ complex, to have enhanced activity in oxidative deformylation reactions believed to involve FeO2+. This mutant showed both decreased N,N-dimethylaniline N-demethylation and N-oxygenation activity. Although some contribution of an FeO2+ species to these reactions cannot be ruled out, formation of product in the iodosylbenzene-supported systems cannot be readily explained by an obligatory FeO2 mechanism and the involvement of FeO3+ is concluded to be more likely.