Salmonella enterica strains survive and propagate in macrophages by both circumventing and resisting the antibacterial effectors normally delivered to the phagosome. An important aspect of Salmonella resistance is the production of periplasmic superoxide dismutase to combat phagocytic superoxide. S. enterica serovar Typhimurium strain 14028 produces two periplasmic superoxide dismutases: SodCI and SodCII. Both enzymes are produced during infection, but only SodCI contributes to virulence in the animal. Although 60% identical to SodCII at the amino acid level with very similar enzymatic properties, SodCI is dimeric, protease resistant, and tethered within the periplasm via a noncovalent interaction. In contrast, SodCII is monomeric and protease sensitive and is released from the periplasm normally by osmotic shock. We have constructed an enzymatically active monomeric SodCI enzyme by site-directed mutagenesis. The resulting protein was released by osmotic shock and sensitive to protease and could not complement the loss of wild-type dimeric SodCI during infection. To distinguish which property is most critical during infection, we cloned and characterized related SodC proteins from a variety of bacteria. Brucella abortus SodC was monomeric and released by osmotic shock but was protease resistant and could complement SodCI in the animal. These data suggest that protease resistance is a critical property that allows SodCI to function in the harsh environment of the phagosome to combat phagocytic superoxide. We propose a model to account for the various properties of SodCI and how they contribute to bacterial survival in the phagosome.Cu/Zn superoxide dismutases (SODs) are metalloproteins that dismute toxic superoxide radicals to H 2 O 2 and O 2 through the alternate oxidation and reduction of the copper(II) ion in the active site. Among bacteria, Cu/Zn SODs (referred to as SodCs) are located in the periplasms of certain gram-negative bacteria (2, 25) and anchored to the surfaces of some grampositive bacteria (13). Salmonella enterica serovar Typhimurium strain 14028 produces two Cu/Zn SODs-SodCI and SodCII. SodCII is the ortholog of Escherichia coli SodC, while SodCI is encoded by the Gifsy-2 prophage. The ability of these two enzymes to contribute to virulence has been studied extensively (12,14,15,24,34,36). Uzzau et al. (36) showed that only SodCI is required for the full virulence of serovar Typhimurium and that SodCII does not contribute to virulence even in the absence of SodCI. Our previous work has confirmed this result, and we have shown that this disparity in virulence phenotypes is due mainly to some differences at the protein level rather than in the regulation of the genes (24). Indeed, SodCI under the control of the more weakly in vivo-induced sodCII promoter (20) was fully capable of complementing wild-type SodCI in an animal infection. These and other data suggested that any differences in enzymatic activity or stability of the active site were insufficient to explain the differential roles of SodC...