Hemoglobin (Hb) from rainbow trout (Oncorhynchus mykiss), Atlantic cod (Gadus morhua), and herring (Clupea harengus) were evaluated in terms of capacity to generate hydroperoxide-derived free radicals in aqueous solution at pH 6.8. Cumene hydroperoxide (CumOOH) was used as a model for preformed lipid hydroperoxides, and free radicals were monitored by stabilizing with the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) and further detection by electron spin resonance spectroscopy. The generation of hydroperoxide-derived free radicals was found to be Hb concentration-dependent, and intermediate Hb concentrations corresponding to hydroperoxide/Hb molar ratios of roughly 200 were found most active. The results indicated the following relative activity in the formation of hydroperoxide-derived free radicals under low and medium hydroperoxide levels (hydroperoxide/Hb molar ratios trout Hb. This free radical generating-activity was not affected by heating fish hemoglobins at 70 degrees C for 10 and 45 min in aqueous solution. Cod and herring Hb also exhibited faster autoxidation rates and stronger redox instability in the presence of hydroperoxides compared to trout Hb. According to their ability to generate hydroperoxide-derived free radicals, hemoglobin from cod and herring showed more activity than trout Hb as catalysts of lipid oxidation in lecithin liposomes. These data emphasize the central contribution of the free radicals formed through hydroperoxide-decomposition on the hemoglobin-mediated lipid oxidation.