The hydroperoxide-supported N-demethylation reactions catalyzed by horseradish peroxidase have been characterized in detail. The ethyl hydroperoxide-supported N-demethylation of N,N-dimethylaniline by horseradish peroxidase resulted in the formation of equimolar amounts of N-methylaniline and formaldehyde with no other products detectable by high performance liquid chromatography analysis of the reaction mixture. One molecule of ethyl hydroperoxide was consumed for each molecule of formaldehyde formed in the reaction. Similar results were obtained for the hydrogen peroxide-supported N-demethylation of N,N-dimethylaniline. The horseradish peroxidase-catalyzed N-demethylation reaction could be supported by a variety of hydroperoxides, peroxides, and peracids. The turnover number for the hydrogen peroxide-supported demethylation reaction (7061) was larger than that for the ethyl hydroperoxide-supported reaction (5382) or for chloroperoxidase- or cytochrome P-450-catalyzed dealkylations. The demethylation reaction exhibited normal Michaelis-Menten saturation kinetics with respect to N,N-dimethylaniline (Km = 0.34 mM) and ethyl hydroperoxide (Km = 0.020 mM), as well as hydrogen peroxide (Km = 0.016 mM). The horseradish peroxidase-catalyzed N-demethylation reaction was not significantly inhibited by reagents which react with the superoxide anion, the hydroxyl radical, or singlet oxygen, suggesting that these activated oxygen species are not free intermediates in the reaction. There was no significant inhibition of the reaction by alpha-phenyl-t-butylnitrone, 5,5-dimethylpyrroline-N-oxide, or other free radical trapping agents. Substitution of D2O for H2O resulted in an inhibition of the reaction with a solvent isotope effect (VH2O/VD2O) of 1.6. Horseradish peroxidase did not catalyze the demethylation of N,N-dimethylaniline-N-oxide, indicating that the reaction does not proceed via N-oxidation of the amine. When the concentrations of both N,N-dimethylaniline and ethyl hydroperoxide were varied in a constant ratio a linear double reciprocal plot was obtained, which is consistent with a ping-pong kinetic mechanism for the horseradish peroxidase-catalyzed demethylation reaction.