To clarify the nature of cytocidal molecular species among the radicals generated in the iron-catalyzed reactions of peroxides (ROOH), we examined the cytocidal effects of these radicals against gram-positive and gram-negative bacteria in the presence or absence of various radical scavengers. Three organic peroxides, t-butyl hydroperoxide (t-BuOOH), methyl ethyl ketone peroxide (MEKOOH), and cumene hydroperoxide, were used. Each radical generated from these peroxides was identified and quantitated by electron spin resonance (ESR) spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The major cytotoxic radical species generated in the mixtures of various peroxides and heme iron, especially methemoglobin, metmyoglobin, or hemin, was the alkyl peroxyl radical (ROO.). Strong bactericidal action against gram-positive bacteria was observed in the peroxide-heme iron system, especially in the case of t-BuOOH and MEKOOH. Killing curves for gram-positive bacteria showed an initial lag period, which may indicate the multihit/multitarget kinetics of cell killing. When the diethylenetriamine pentaacetic acid (DTPA)-Fe2+ complex was used as a catalyst for decomposition of various peroxides, alkyl, alkoxyl, and alkyl peroxyl radicals were identified by spin-trapping analysis. However, study of the time course of alkyl peroxyl radical production in the DTPA-Fe2+ complex system revealed that radical species generated in this system were very short lived: a maximal level was achieved within 1 min and then declined sharply, and no bactericidal activity was observed after 10 min. In contrast, the alkyl peroxyl radical level generated by the organic peroxide-heme iron system remained high for 30 min or longer. The generation of alkyl peroxyl radicals quantified by ESR correlated quite well with the bactericidal effect of the system of peroxide plus iron. In addition, bactericidal activity was completely inhibited by the addition of the spin trap DMPO, as well as of other various radical scavengers (alpha-tocopherol and L-ascorbic acid), into the peroxide-heme iron system, but this effect was not observed with superoxide dismutase, beta-carotene, dimethyl sulfoxide, diphenylamine, or butylated hydroxyltoluene. In view of these results, it is assumed that alkyl peroxyl radicals are the potent molecular species that are cytotoxic against bacteria, whereas alkoxyl radicals (RO.) generated in this system do not affect bacterial viability.