Spectroscopic and kinetic investigations have been carried out on catalase from bovine liver and from Aspergillus niger to address the mechanism of activity loss at high hydrogen peroxide concentrations (0.01 to 2 M). The mammalian enzyme was both reversibly inhibited and irreversibly inactivated in the presence of hydrogen peroxide, whereas the fungal enzyme did not show any reversible inhibition. A comparison of reaction rates with catalase preparations containing different proportions of Compound III indicated that the formation of Compound III is responsible for the reversible inhibition of bovine liver catalase at high H2O2 concentrations. Superoxide radical did not appear to be the inactivating species in this mechanism. Kinetic modelling emphasises the role of Compound III in both types of activity loss. It shows that the higher activity of A. niger catalase at high substrate concentration, compared to bovine liver catalase, the lack of reversible inhibition of the former and its lower rate of irreversible inactivation may be attributed both to a high rate of conversion of Compound III into native form and to a low rate of conversion of Compound I to Compound II.