Francisella tularensis, the highly virulent etiologic agent of tularemia, is a low-dose intracellular pathogen that is able to escape from the phagosome and replicate in the cytosol. Although there has been progress in identifying loci involved in the pathogenicity of this organism, analysis of the genome sequence has revealed few obvious virulence factors. We previously reported isolation of an F. tularensis subsp. tularensis strain Schu S4 transposon insertion mutant with a mutation in a predicted hypothetical lipoprotein, FTT1103, that was deficient in intracellular replication in HepG2 cells. In this study, a mutant with a defined nonpolar deletion in FTT1103 was created, and its phenotype, virulence, and vaccine potential were characterized. A phagosomal integrity assay and lysosome-associated membrane protein 1 colocalization revealed that DeltaFTT1103 mutant bacteria were defective in phagosomal escape. FTT1103 mutant bacteria were maximally attenuated in the mouse model; mice survived, without visible signs of illness, challenge by more than 10(10) CFU when the intranasal route was used and challenge by 10(6) CFU when the intraperitoneal, subcutaneous, or intravenous route was used. The FTT1103 mutant bacteria exhibited dissemination defects. Mice that were infected by the intranasal route had low levels of bacteria in their livers and spleens, and these bacteria were cleared by 3 days postinfection. Mutant bacteria inoculated by the subcutaneous route failed to disseminate to the lungs. BALB/c or C57BL/6 mice that were intranasally vaccinated with 10(8) CFU of FTT1103 mutant bacteria were protected against subsequent challenge with wild-type strain Schu S4. These experiments identified the FTT1103 protein as an essential virulence factor and also demonstrated the feasibility of creating defined attenuated vaccines based on a type A strain.