Inactivation of horseradish peroxidase (HRP) was examined in the presence of hydrogen peroxide alone and in the presence of hydrogen peroxide plus phenol. HRP is inactivated upon exposure to hydrogen peroxide (H2O2) by the combination of two possible pathways, dependent upon hydrogen peroxide concentration. At low H2O2 concentrations (below 1.0 mM in the absence of phenol), inactivation is predominantly reversible, resulting from the formation and accumulation of catalytically inert intermediate compound III. As H2O2 concentrations increase, an irreversible mechanism-based inactivation process becomes predominant. The overall inactivation comprised of both processes exhibits a second-order inactivation rate constant (kapp) of 0.023 +/- 0.005 M-1 s-1 at pH 7.4 and 25 degrees C. In the presence of both hydrogen peroxide fixed at 0.5 mM and phenol, HRP was inactivated in an irreversible, time- and phenol concentration-dependent process, also mechanism-based, with a kapp of 0.019 +/- 0.004 M-1 s-1.