A supernatant factor prepared from 48-h cultures of Salmonella typhimurium has been used to immunize mice against subsequent challenge with normally lethal doses of S. typhimurium. The mouse strains used, C57BL and BALB/c, were sensitive to S. typhimurium with 50% lethal doses of less than 50 organisms. Two doses of supernatant factor, given intraperitoneally 20 days apart, protected mice against a subcutaneous challenge dose 10 days later of 100 50% lethal doses of S. typhimurium, resulting in 50 to 80% survival. The viable counts were reduced initially in organs of immunized mice compared with controls, and the multiplication of bacteria was delayed, although the final levels found in the organs would normally have been lethal. Protection obtained was specific for S. typhimurium in that no increased survival was shown after Salmonella enteritidis challenge of immunized mice. Although lipopolysaccharide was demonstrated in the supernatant factor, lipopolysaccharide alone did not protect challenged mice. Supernatant factor produced delayed-type hypersensitivity reactions in mice sensitized with nonlethal doses of Salmonella. The nature of the active factor, found to be partially protein, has yet to be elucidated.