The effects of high-pressure treatment on the reaction rates of horseradish peroxidase (HRP) with guaethol or guaiacol as a hydrogen donor were evaluated from direct transmission measurements in a high-pressure optical cell. at 435 nm. Peroxidases are known to be very barostable, and insensitive to heat. With guaethol the reaction velocity was independent of pressure up to 500 MPa, but with guaiacol the cytochrome c oxidase underwent a mechanism-based irreversible inhibition of catalytic activity when subjected to pressure; in the resting states (fully oxidized or reduced), it was insensitive to pressure. The enzyme inactivation took place with an inactivation rate constant of 5.15 x 10(-1) min(-1) at 500 MPa, 25 degreesC and pH 7. The degree of inactivation was correlated to the concentration of guaiacol. This is the first report on a mechanism-based pressure inactivation of HRP triggered at moderate pressure and temperature and mediated by the hydrogen donor.