The involvement of nitric oxide (NO) in host defense and cytoprotective functions in murine salmonellosis has been reported. Salmonella mutants with the altered sigma factor RpoS (sigmaS) are less virulent and are susceptible to various stresses. This study investigated the role of the rpoS gene of Salmonella enterica serovar Typhi in NO-dependent host defense in vitro and in vivo. Wild-type mice and mice deficient in inducible NO synthase (iNOS) were infected intraperitoneally or orally with serovar Typhi strains. iNOS-deficient mice were more susceptible to infection by both wild-type and rpoS mutant strains of serovar Typhi and showed extensive apoptotic liver damage compared with wild-type mice. Intracellular killing of Salmonella was analyzed with RAW 264 macrophage-like cells and primary peritoneal macrophages from wild-type and iNOS-deficient mice after cells were infected with the serovar Typhi parent or rpoS mutant strain. The rpoS mutant was more susceptible to killing by macrophages than was the wild-type strain. Also, the wild-type strain produced more extensive apoptotic changes in macrophages than did rpoS mutant. These effects were nullified in RAW 264 cells treated with an NOS inhibitor and in iNOS-deficient primary macrophages. Peroxynitrite susceptibility assays of these strains were also performed. The rpoS mutant Typhi strain was more sensitive to in vitro peroxynitrite treatment than was the parent strain. Together these data show that NO has a significant host defense function during serovar Typhi infection, and that Salmonella RpoS, because it reacts to the presence of NO or its reactive derivatives, is thought to have a role in the pathogenicity of serovar Typhi.