Francisella tularensis gives rise to two distinct colony types, acriflavine agglutination test-positive (acf⁺) and -negative (acf^-) colonies. The acf⁺ variants were exclusively low virulent in mice, while the acf^- variants were shown to be either high or low virulent. Three fractions, phosphate-buffered saline-extractable without heating, with heating at 60C, and with heating at 100C, were obtained from cultures of both the acf⁺ and acf^- variants on agar media, and the polysaccharide antigens in those fractions were quantitated. All of the highly virulent acf^- variants possessed a large amount of the polysaccharide antigen in the fraction extractable with heating at 60C. This antigen was not, however, detected in any of the acf⁺ variants and one low-virulent acf^- variant. It was also detected in a very low amount in some other acf^- variants with low virulence. The amount of this polysaccharide antigen was therefore shown to be correlated with bacterial virulence in mice.