Francisella tularensis is the bacterial cause of tularemia and has been designated as a potential bioweapon due to its high infectivity and virulence. F. tularensis infects the liver of its mammalian hosts and replicates in hepatocytes, the major cells of the liver, both in vivo and in vitro. However, the factors that govern adaptation of F. tularensis to the intra-hepatocytic niche have not been identified. Using cDNA microarrays, we determined the transcriptional profile of the live vaccine strain (LVS) of F. tularensis growing in the FL83B murine hepatocytic cell line. Expression of 53 genes was up-regulated more than 2-fold compared with organisms cultured in broth. The fslC gene of the fsl operon was the most highly up-regulated (about 13-fold). In-frame deletion of fslC eliminated the ability of the LVS to produce siderophores, which are involved in uptake of ferric iron, and inhibited growth of the bacterium in iron-restricted media. In-frame deletion of feoB, which encodes a putative bacterial ferrous iron transporter, also retarded replication of the LVS in iron-restricted media. Growth of the Ç_fslC and Ç_feoB mutants was the same as the wild-type LVS in human monocyte-derived macrophages. In FL83B hepatocytic cells, replication of the Ç_feoB strain was diminished, whereas growth of the Ç_fslC mutant was normal. However, growth of both mutants was inhibited in hepatocytes depleted of intracellular iron. Furthermore, the virulence of both mutant strains was attenuated in mice infected intranasally. FeoB thus represents a previously unidentified pathway for uptake of ferrous iron in F. tularensis, and our results show that both FeoB and FslC contribute to virulence of the bacterium. Microarray analysis also was performed to determine how hepatocytes respond to infection with the F. tularensis LVS. Murine AML12 hepatocytes infected in vitro up-regulated expression of genes encoding proinflammatory cytokines, chemokines, colony-stimulating factors (CSF), and an adhesion molecule that binds immune cells. Enhanced production of CSF-3 and the chemokines CXCL2 and CCL20 was verified at the protein level by immunoassay. These results suggest that hepatocytes may play an important role in host defense against F. tularensis by recruiting immune cells to foci of infection.