The mechanism of activation of Escherichia coli redox sensory protein SoxR is still unclear: a [2Fe-2S] cluster contained in a SoxR dimer is potentially redox-sensitive, but the nature of the signal is unknown. Antioxidant vitamins C (ascorbate) and E (alpha-tocopherol) were used to explore the mechanism of activation of the SoxR protein in vivo. Treating E. coli cells with ascorbate or alpha-tocopherol increased their tolerance to paraquat (PQ, a redox-cycling compound), even in the absence of the soxRS locus, suggesting a radical-quenching activity. When using a soxS'::lacZ fusion, whose expression is governed by activated SoxR, ascorbate and alpha-tocopherol also prevented the expression of beta-galactosidase after PQ treatment. A secondary activity was observed in cells carrying soxR101, a mutation resulting in the constitutive expression of the sox regulon, where the overexpression of soxS'::lacZ was also reduced by ascorbate or alpha-tocopherol treatment. Additionally, different mechanisms of action were revealed as alpha-tocopherol was capable of preventing both PQ and meanadione (MD) lethality, whilst ascorbate prevented PQ lethality but increased MD-mediated cell death. It is proposed that alpha-tocopherol, positioned in membranes, can prevent superoxide-dependent membrane damage; however, water-soluble ascorbate is unable to do so and can even increase the concentration of oxygen radicals reacting with released membrane-associated Fe(II).