The ferritins are a class of iron storage and detoxification proteins that play a central role in the biological management of iron. These proteins have a catalytic site, "the ferroxidase site", located on the H-type subunit that facilitates the oxidation of Fe(II) to Fe(III) by O(2). Measurements during the past 10 years on a number of vertebrate ferritins have provided evidence that H(2)O(2) is produced at this diiron ferroxidase site. Recently reported experiments using three different analytical methods with horse spleen ferritin (HoSF) have failed to detect H(2)O(2) production in this protein [Lindsay, S., Brosnahan, D., and Watt, G. D. (2001) Biochemistry 40, 3340-3347]. These findings contrast with earlier results reporting H(2)O(2) production in HoSF [Xu, B., and Chasteen, N. D. (1991) J. Biol. Chem. 266, 19965-19970]. Here a sensitive fluorescence assay and an assay based on O(2) evolution in the presence of catalase were used to demonstrate that H(2)O(2) is produced in HoSF as previously reported. However, because of the relatively few H-chain ferroxidase sites in HoSF and the reaction of H(2)O(2) with the protein, H(2)O(2) is more difficult to detect in this ferritin than in recombinant human H-chain ferritin (HuHF). The proper sequence of addition of reagents is important for measurement of the total amount of H(2)O(2) produced during the ferroxidation reaction.