The nature of the stress experienced by Escherichia coli K-12 exposed to chromate, and mechanisms that may enable cells to withstand this stress, were examined. Cells that had been preadapted by overnight growth in the presence of chromate were less stressed than nonadapted controls. Within 3 h of chromate exposure, the latter ceased growth and exhibited extreme filamentous morphology; by 5 h there was partial recovery with restoration of relatively normal cell morphology. In contrast, preadapted cells were less drastically affected in their morphology and growth. Cellular oxidative stress, as monitored by use of an H2O2-responsive fluorescent dye, was most severe in the nonadapted cells at 3 h postinoculation, lower in the partially recovered cells at 5 h postinoculation, and lower still in the preadapted cells. Chromate exposure depleted cellular levels of reduced glutathione and other free thiols to a greater extent in nonadapted than preadapted cells. In both cell types, the SOS response was activated, and levels of proteins such as SodB and CysK, which can counter oxidative stress, were increased. Some mutants missing antioxidant proteins (SodB, CysK, YieF, or KatE) were more sensitive to chromate. Thus, oxidative stress plays a major role in chromate toxicity in vivo, and cellular defense against this toxicity involves activation of antioxidant mechanisms. As bacterial chromate bioremediation is limited by the toxicity of chromate, minimizing oxidative stress during bacterial chromate reduction and bolstering the capacity of these organisms to deal with this stress will improve their effectiveness in chromate bioremediation.