Electron spin resonance (ESR) spectroscopy and the spin-trapping technique were used to investigate the capacity of several hemoglobin (Hb) forms of rainbow trout (oxyHb and metHb), free hemin (oxidized form of heme group), and hemin complexed with bovine serum albumin (BSA) to promote formation of free radicals via fragmentation of preformed lipid hydroperoxides. Cumene hydroperoxide (CumOOH) was used as a model for lipid hydroperoxide, and free radicals were monitored by stabilizing with the spin traps alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Two different types of free radicals, hydroxyl and carbon-centered radicals, were identified as a result of the interaction of the heme-containing systems and CumOOH. Carbon-centered radicals were found to be mainly heme-mediated because the addition of the iron-chelating agent EDTA did not affect the formation of POBN/carbon-centered adducts. Hemin alone was the best promoter for the production of POBN/carbon-centered radicals in the presence of low hydroperoxide concentrations (below equimolar condition over heme group), whereas hemin/BSA and oxyHb were more active in generating radicals at high hydroperoxide concentrations or after successive interactions with hydroperoxides. This finding can be explained by the coexistence of two different facts: (i) the interaction between hemin and lipid hydroperoxides seems to be more efficient in the case of free hemin compared to heme-protein complexes and (ii) a faster degradation of hemin is produced without the presence of a protein fraction, globin or albumin. The comparison of oxyHb and metHb also suggested that both Hb redox states have similar capacities to generate oxidative stress via cleavage of preformed lipid hydroperoxides.