Abstract: A scan of F. tularensis genome for homology to a regulatory protein that controls virulence identified gene FTL0552. A knock out mutation in FTL0552 was created using reverse transcriptase PCR and the construct inserted into F. tularensis. This mutant was defective for survival in macrophages and found avirulent in in vivo testing, where the mutant exhibited reduced levels of pro-inflammatory cytokine production, reduced evidence of histopathology in affected tissues, reduced systemic infection, and rapid clearance of the bacterium. In vivo challenge studies with the FTL0552 mutant using the virulent F. tularensis subsp. tularensis SchuS4 strain show an immune response is induced, and protection afforded, after preexposure to the FTL0552 mutant. Microarray studies revealed 148 genes regulated by FTL0552, including genes located within the FPI that are essential for intracellular survival.