It has been found previously that vitamin B12b amplifies significantly the cytotoxic effects of ascorbic acid by catalyzing the formation of reactive oxygen species, and the antioxidant dithiothreitol (DTT), in contrast to catalase, does not prevent the cytotoxicity. Therefore, in this study we examined whether B12b is able to enhance the cytotoxicity of DTT. It was revealed that B12b strongly increases the cytotoxic effect of DTT. Vitamin B12b added to DTT catalyzed the generation and drastic accumulation of hydrogen peroxide in culture medium to a concentration of 260 microM within 7 min. The extracellular oxidative burst induced by the combination of B12b and DTT (DTT + B12b) was accompanied by intracellular oxidative stress, the destabilization of lysosomes, and damage to DNA. The accumulation of DNA lesions led to the initiation of apoptotic cell death, including the activation of caspase-3 and the release of cytochrome c. The antioxidants pyruvate and catalase completely prevented the DTT + B12b-induced oxidative stress and cell death. The iron chelators desferrioxamine and phenanthroline prevented the geno- and cytotoxic action of the combination although they did not reduce the exogenous oxidative burst, indicating a key role for intracellular iron in the cytotoxicity of the combination. Thus, vitamin B12b dramatically enhances the cytotoxicity of DTT, catalyzing the generation of hydrogen peroxide and inducing extra- and intracellular oxidative stress, early destabilization of lysosomes, and iron-dependent DNA damage.