Metabolic activation of peroxides and hydroperoxides to free radicals is associated with the tumor promoting activity of these compounds. tert-Butyl hydroperoxide (t-BOOH) metabolism has been extensively studied as a model of peroxide biotransformation. In vivo studies are limited, and the hemoglobin-thiyl radical was the only species thus far identified in the blood of treated rats. Here we further examine t-BOOH metabolism in vivo with regard to free radical and DNA adduct production. Spin-trapping experiments with phenyl-N-tert-butylnitrone (PBN) led to the detection of EPR signals in the blood, bile, and organic extracts of the liver and stomach of rats treated with t-BOOH. Analysis of these signals demonstrated that t-BOOH metabolism in vivo produces alkyl radicals, detected in the bile and organic extracts of liver and stomach, in addition to the previously identified hemoglobin-thiyl radical. To characterize the produced alkyl radicals, experiments were performed with (13)C-labeled t-BOOH and two spin traps, PBN and alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN). The latter was used because the EPR signals obtained with PBN were too weak to be unambiguous. Nevertheless, the EPR signals present in the bile of animals treated with (13)C-labeled t-BOOH and PBN or POBN were consistent with adducts of (13)C-labeled methyl radical and an unidentified alkyl radical. The latter is probably derived from lipids oxidized by the metabolically produced primary radicals, methyl and its precursor, tert-butoxyl. The presence of 8-methylguanine and 7-methylguanine in hydrolysates of DNA from liver and stomach of rats treated with t-BOOH was also examined. 8-Methylguanine, a typical product of methyl radical attack on DNA, was detectable in both the liver and stomach of treated rats. The results may be relevant to the understanding of the genotoxic properties of other peroxides, particularly of cumene hydroperoxide.