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The intracellular pathogen, Salmonella enterica serovar Typhimurium, is able to proliferate in phagocytes, although reactive oxygen and nitrogen intermediates are lethal to most phagocytosed bacteria. To determine whether repair of oxidatively damaged DNA is involved in S. typhimurium intramacrophage proliferation, null mutants of the DNA base excision repair (BER) system were generated. These mutants were deficient in discrete enzymes (Deltanth, Deltanei, Deltaxth, Deltanfo) or in the defined glycosylase (Deltanth/nei) and endonuclease (Deltaxth/nfo) steps. In this study, S. typhimurium BER mutants are characterized for the first time. In vitro characterization of the Salmonella BER mutants revealed phenotypes that are mostly consistent with characterized Escherichia coli BER mutants. These strains were used to evaluate the role of BER in the context of Salmonella virulence. S. typhimurium Deltaxth and Deltaxth/nfo were significantly impaired for survival in both cultured and primary macrophages activated with interferon (IFN)-gamma. Survival of Deltaxth and Deltaxth/nfo was improved nearly to wild-type levels in activated primary macrophages lacking both phagocyte oxidase and inducible nitric oxide synthase. In the murine typhoid fever model, Deltanth/nei was fivefold attenuated and Deltaxth/nfo was 12-fold attenuated compared with wild type. These data indicate that DNA oxidation is a mechanism that macrophages use to damage intracellular Salmonella, and suggest that BER-mediated repair of this damage may be important in the establishment of Salmonella infection. We speculate that adaptation to a pathogenic lifestyle may influence the acquisition and retention of redundant BER enzymes.