Superoxide is a key component of the antibacterial weaponry of phagocytes. Presumably, for this reason, strains of Salmonella typhimurium express a periplasmic superoxide dismutase (SOD) that is essential for full virulence. Because most anions cannot easily penetrate lipid membranes, it is thought that the phagosomal superoxide either damages an unknown target on the bacterial surface or reacts with nitric oxide to form peroxynitrite (HOONO), a toxic oxidant that can freely enter bacteria. However, in this study, we tested whether superoxide itself could penetrate membranes. Superoxide that was generated at high pH (>7.5) very slowly reduced cytochrome c that was encapsulated inside lipid vesicles. It did so much more quickly at lower pH (<7). Under the latter conditions, more superoxide was protonated and uncharged (HO2*), and the penetrance of superoxide was proportional to the concentration of this species. The permeability coefficient of HO2* was determined to be 9 x 10(-4) cm sec(-1), just slightly lower than that of water and far higher than the value of the anionic form (O2-, <10(-7) cm sec(-1). When Escherichia coli mutants that lack periplasmic SOD were exposed to super-oxide at pH 6.5, cytosolic fumarase B was damaged. Damage was minimal at higher pH or in strains that contained periplasmic SOD. Thus, in the acidic phagolysosome, superoxide may be able to penetrate and attack cytosolic targets of captive bacteria. This process may contribute to the potency of the oxidative burst. One role of periplasmic SOD may be to avert this damage. In contrast, periplasmic SOD was ineffective at lowering the extracellular super-oxide concentration and, therefore, may have little impact upon HOONO formation.