A gene cluster encoding enzymes involved in LPS O antigen biosynthesis was identified from the partial genome sequence of Francisella tularensis subsp. tularensis Schu S4. All of the genes within the cluster were assigned putative functions based on sequence similarity with genes from O antigen biosynthetic clusters from other bacteria. Ten pairs of overlapping primers were designed to amplify the O antigen biosynthetic cluster by PCR from nine strains of F. tularensis. Although the gene cluster was present in all strains, there was a size difference in one of the PCR products between subsp. tularensis strains and subsp. holarctica strains. LPS was purified from F. tularensis subsp. tularensis Schu S4 and the O antigen was shown by mass spectrometry to have a structure similar to that of F. tularensis subsp. holarctica strain 15. When LPS from F. tularensis subsp. tularensis Schu S4 was used to immunize mice that were then challenged with F. tularensis subsp. tularensis Schu S4, an extended time to death was observed.