The alternative sigma factor RpoS (sigma S) is required for Salmonella virulence in mice. We report the immunizing capacity of Salmonella typhimurium rpoS and rpoS aroA mutants to protect susceptible BALB/c mice against subsequent oral challenge with virulent S. typhimurium. When administered orally or intraperitoneally, rpoS derivatives of the mouse-virulent S. typhimurium strains, C52 and SL1344, were highly attenuated and were efficient single-dose live vaccines. rpoS aroA mutants were more attenuated than corresponding single aroA or rpoS mutants, as assessed after oral or intraperitoneal administration, but retained significant ability to protect mice against salmonellosis. Salmonella rpoS and rpoS aroA mutants therefore deserve serious consideration for rational vaccine design. Consistent with this, Salmonella typhi Ty2, a 'wild-type' strain used widely for the development of human live-vaccine candidates against typhoid fever, was shown to be defective for rpoS. In addition, our results demonstrate that rpoS not only controls the growth and persistence of S. typhimurium in deep lymphoid organs, but also plays a role during the initial stages of oral infection.