The o-quinone forms of 2,3- and 3,4-catechol estrogens have been implicated in the carcinogenicity of these hormones. The concomitant production of reactive oxygen species during reduction of the o-quinone estrogens has been inferred to play a mechanistic role in their mutagenic potential. Conclusive evidence documenting the production of hydrogen peroxide, the hydroxyl radical, and the estrone 3,4-semiquinone in estrone 3,4-quinone (3,4-EQ)-treated human breast cancer subcellular fractions was demonstrated in the absence of exogenously added catalysts. Subcellular fractions of MCF-7 cells treated with 3,4-EQ and NADPH, including nuclei, mitochondria, and microsomes, were shown to support significant amounts of hydrogen peroxide production. Hydrogen peroxide production in 3,4-EQ-treated cellular fractions and the chromosomal DNA damage induced in 3,4-EQ-treated MCF-7 cells were abolished by the addition of catalase. A significant and potentially physiologically relevant spontaneous reduction of 3,4-EQ by NADPH resulting in hydrogen peroxide production was demonstrated. The results unequivocally demonstrate that free radicals are produced during the metabolism of estrone 3,4-quinone in human cells.