Correlation between the virulence of Francisella tularensis in experimental mice and its acriflavine reaction was studied. The cultures derived from all four strains (Ebina, CMB2, Schu, and N9) that had long been subcultured on agar media yielded two types of colonies, i.e., acriflavine reaction-positive (acf+) and acriflavine reaction-negative (acf-) colonies. All acf+ colonies, regardless of their parent strains, were shown to be low virulent in mice. Acf -colonies were shown to be either high (Ebina, CM B2) or low (Schu, N9) virulent. The low-virulent acf-colonies gained virulence during several passages in mice, whereas the acf+ colonies remained low virulent even after the animal passages,