The effect of butylated hydroxytoluene (BHT) and its metabolites on DNA cleavage in vitro was studied with supercoiled plasmid DNA, pUC18, by agarose gel electrophoresis. Among several BHT metabolites, 2,6-di -t-butyl-p-benzoquinone (BHT-quinone) caused cleavage of supercoiled DNA (form I) at a concentration as low as 1 × 10−6 M. The relative amount of linear form (form III) was increased with increasing concentration of BHT-quinone. 2,6-Di-t-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BHT-peroxyquinol) and 3,5-di-t-butyl-4-hydroxybenzaldehyde (BHT-CHO) also cleaved DNA, but to a lesser extent than BHT-quinone. No DNA cleavage was detected by BHT, 2,6-di-t-butyl-4-hydroxymethyl phenol (BHT-OH), 3,5-di-t-butyl-4-hydroxybenzoic acid (BHT-COOH), 2,6-di-t-butyl-4-hydroxy-4-methyl-2,5-cyclohexadienone (BHT-quinol) or 2,6-di-t-butyl-4-methylene-2,5-cyclohexadienone (BHT-quinone methide). The DNA cleavage by BHT-quinone was inhibited by oxygen radical scavengers including Superoxide dismutase (SOD), catalase, polyethylene glycol,t-butyl alcohol, dimethyl sulfoxide, sodium azide, sodium benzoate, bovine serum albumin and methionine, while it was enhanced by the addition of FeCl2. The production of Superoxide radical in a solution of BHT-quinone was confirmed by cytochrome c reduction assay. Superoxide was not produced by BHT or other BHT metabolites except for BHT-quinone. These results suggest that BHT-quinone, one of the principal metabolites of BHT, cleaves DNA strands via its generation of oxygen radicals. Such modification of DNA observed in vitro may be relevant to genotoxicity by BHT after metabolic activation in vivo.