The effects of temperature (20 to -38 degrees C), pressure (normal pressures to 1.2 kbar) and solvent (water, 60% DMSO and 50% methanol) on the reaction of hydrogen peroxide or ethyl peroxide with horseradish peroxidase were studied. The formation of compound I was followed at 403 nm in a stopped flow apparatus adapted for high pressure and low temperature work. As with the alkaline form (Job and Dunford 1978), the neutral form of the peroxidase binds peroxide substrates in two steps. It was the combined use of organic solvents and low temperatures which revealed saturation kinetics: (Formula: see text) compound I, where E = horseradish peroxidase and S peroxide substrate. In water and organic solvents at temperatures above -10 degrees C, K1 was too small and k2 too large to be measured, here K1 X k2 was obtained. k-2 was too small for measurement under all conditions. Whereas K1 was insensitive to the peroxide substrate and solvent composition, k2 was very sensitive. The thermodynamic parameters delta H, delta S and delta V for K1 and k2 were obtained under different experimental conditions and the data are interpreted within the available thermodynamic theories.