Cells devoid of cytosolic superoxide dismutase (SOD) suffer enzyme inactivation, growth deficiencies, and DNA damage. It has been proposed that the scant superoxide (O2-) generated by aerobic metabolism harms even cells that contain abundant SOD. However, this idea has been difficult to test. To determine the amount of O2- that is needed to cause these defects, we modulated the O2- concentration inside Escherichia coli by controlling the expression of SOD. An increase in O2- of more than twofold above wild-type levels substantially diminished the activity of labile dehydratases, an increase in O2- of any more than fourfold measurably impaired growth, and a fivefold increase in O2- sensitized cells to DNA damage. These results indicate that E. coli constitutively synthesizes just enough SOD to defend biomolecules against endogenous O2- so that modest increases in O2- concentration diminish cell fitness. This conclusion is in excellent agreement with quantitative predictions based upon previously determined rates of intracellular O2- production, O2- dismutation, dehydratase inactivation, and enzyme repair. The vulnerability of bacteria to increased intracellular O2- explains the widespread use of superoxide-producing drugs as bactericidal weapons in nature. E. coli responds to such drugs by inducing the SoxRS regulon, which positively regulates synthesis of SOD and other defensive proteins. However, even toxic amounts of endogenous O2- did not activate SoxR, and SoxR activation by paraquat was not at all inhibited by excess SOD. Therefore, in responding to redox-cycling drugs, SoxR senses some signal other than O2-.