1. Mitochondrial H2O2 formation is not in equilibrium with defence mechanisms that counteract an accumulation of H2O2 in rat-heart cells. 2. A model for the accumulation kinetics is proposed which is consistent with the data presented. 3. Four different pathways of H2O2 metabolism are described in rat-heart mitochondria. The major site for metabolic branching of H2O2 via different routes was found to be the mitochondrial catalase. 4. Glutathione (GSH) peroxidase accounts for only 15% of intramitochondrial H2O2 metabolism, while catalase-mediated destruction is four times more rapid. 5. Catalase activity is limited by its structural compartmentation in the matrix, while GSH peroxidase activity was found to be dependent on the availability of free GSH. 6. Catalase was shown to protect rat-heart mitochondria from upsetting redox states of GSH and pyridine nucleotides following H2O2 decomposition by GSH peroxidase. 7. Computer simulations of experimental data suggest the existence of a third sink for mitochondrial H2O2, possibly due to mitochondrial formation of OH . radicals; another fraction of the H2O2 matrix pool may cross the mitochondrial membrane and accumulate in the cytosol.