BACKGROUND Francisella tularensis is a zoonotic intracellular bacterial pathogen that causes tularemia. The subspecies tularensis is highly virulent and is classified as a category A agent of biological warfare because of its low infectious dose by an aerosol route, and its ability to cause severe disease. In macrophages F. tularensis exhibits a rather novel intracellular lifestyle; after invasion it remains in a phagosome for three to six hours before escaping to, and replicating in the cytoplasm. The molecular mechanisms that allow F. tularensis to invade and replicate within a host cell have not been well defined. METHODS We constructed a stable transposon mutagenesis library of virulent strain Schu S4 using a derivative of the EZ::TN transposon system. Approximately 2000 mutants were screened for the inability to invade, and replicate in the hepatic carcinoma cell line HepG2. These mutants were also tested for replication within the J774.1 macrophage-like cell line. RESULTS Eighteen mutants defective in intracellular replication in HepG2 cells were identified. Eight of these mutants were auxotrophs; seven had mutations in nucleotide biosynthesis pathways. The remaining mutants had insertions in genes that were predicted to encode putative transporters, enzymes involved in protein modification and turnover, and hypothetical proteins. A time course of the intracellular growth of a pyrB mutant revealed that this mutant was only able to grow at low levels within HepG2 cells but grew like wild-type bacteria in J774.1 cells. This pyrB mutant was also attenuated in mice. CONCLUSIONS This is the first reported large-scale mutagenesis of a type A strain of F. tularensis and the first identification of mutants specifically defective in intracellular growth in a hepatic cell line. We have identified several genes and pathways that are key for the survival and growth of F. tularensis in a hepatic cell line, and a number of novel intracellular growth-defective mutants that have not been previously characterized in other pathogens. Further characterization of these mutants will help provide a better understanding of the pathogenicity of F. tularensis, and may have practical applications as targets for drugs or attenuated vaccines.