The capacity of rat liver homogenates and mitochondria to remove H(2)O(2) was determined by comparing their ability to slow fluorescence generated by a H(2)O(2) 'detector' with that of desferrioxamine solutions. H(2)O(2) was produced by glucose oxidase-catalysed glucose oxidation. The capacity to remove H(2)O(2) was expressed as equivalent concentration of desferrioxamine. The method showed changes in the capacity of H(2)O(2) removal after treatment with ter-butylhydroperoxide or glutathione. The H(2)O(2) removal capacity of homogenates and mitochondria from rat liver, heart, and skeletal muscle was compared with their overall antioxidant capacity. For homogenates, the order of both antioxidant and H(2)O(2) removal capacities was liver>heart>muscle. For mitochondria, the order of the antioxidant capacities mirrored that of the homogenates, while the order of the H(2)O(2) removal capacities was heart>muscle>liver. Because H(2)O(2) removal is not only due to H(2)O(2)-metabolizing enzymes, but also to hemoproteins that convert H(2)O(2) into more reactive radicals via Fenton reaction, the higher concentration of cytochromes in mitochondria of cardiac and skeletal muscles can explain the above discrepancy. A higher H(2)O(2) removal capacity was found to be associated with a higher rate of H(2)O(2) release by mitochondria, indicating that the order of H(2)O(2) release rate mirrors that of H(2)O(2) production rate. We suggest that the different capacities of the mitochondria from the three tissues to produce reactive oxygen species are due to differences in the concentration of respiratory mitochondrial chain components in the reduced form.