A supernatant factor (SF) prepared from cultures of Salmonella typhimurium protected naturally susceptible inbred mice against challenge with S. typhimurium subcutaneously injected (s.c.i.), but not against Salmonella enteritidis, suggesting tha the relevant specificity involved lipopolysaccharide. Further experiments were performed with two transductant strains of S. typhimurium. Strain SH6701 has O-antigen 4 from S. typhimurium and SH6703 had O-antigen 9 from S. enteritidis. Immunization with SF from SH6701 protected 95% BALB/c mice challenged with 100 LD50 S. typhimurium s.c.i., whereas SH6703 immunization had no effect on survival or mean survival time. SH 6703 SF gave some protection against homologous challenge. Antibody titres and delayed-type hypersensitivity reactions were also tested in immunized mice. The SF was, therefore, specific in that O-antigen 4 was necessary to protect mice against S. typhimurium challenge. Since we have previously demonstrated protein to be necessary for protection by SF, the active factor may be in the form of a protein-lipopolysaccharide complex.