A successful transition of Listeria monocytogenes from the extracellular to the intracellular environment requires a precise adaptation response to conditions encountered in the host milieu. Although many key steps in the intracellular lifestyle of this gram-positive pathogen are well characterized, our knowledge about the factors required for cytosolic proliferation is still rather limited. We used DNA microarray and real-time reverse transcriptase PCR analyses to investigate the transcriptional profile of intracellular L. monocytogenes following epithelial cell infection. Approximately 19% of the genes were differentially expressed by at least 1.6-fold relative to their level of transcription when grown in brain heart infusion medium, including genes encoding transporter proteins essential for the uptake of carbon and nitrogen sources, factors involved in anabolic pathways, stress proteins, transcriptional regulators, and proteins of unknown function. To validate the biological relevance of the intracellular gene expression profile, a random mutant library of L. monocytogenes was constructed by insertion-duplication mutagenesis and screened for intracellular-growth-deficient strains. By interfacing the results of both approaches, we provide evidence that L. monocytogenes can use alternative carbon sources like phosphorylated glucose and glycerol and nitrogen sources like ethanolamine during replication in epithelial cells and that the pentose phosphate cycle, but not glycolysis, is the predominant pathway of sugar metabolism in the host environment. Additionally, we show that the synthesis of arginine, isoleucine, leucine, and valine, as well as a species-specific phosphoenolpyruvate-dependent phosphotransferase system, play a major role in the intracellular growth of L. monocytogenes.Listeria monocytogenes is a gram-positive, food-borne bacterium that is widely distributed in nature and can cause serious infection in susceptible individuals (9). The infection pathogenesis of this facultative intracellular pathogen includes phases where the bacteria successfully proliferate in the challenging environment in a variety of mammalian cell types like epithelial cells, endothelial cells, hepatocytes, dendritic cells, and macrophages. While invading the host cells, L. monocytogenes is confronted with suboptimal growth conditions, such as a rapid pH drop within the phagosome (1). Following early escape from the primary phagosome by membrane lysis, the pathogen is released into the cytosol where it encounters conditions of nutrient and iron starvation. The genes essential for the internalization of L. monocytogenes by the mammalian host cells (inlA and inlB), phagosomal escape (hly, plcA, and plcB), and intra-and intercellular motility (actA) have been extensively studied in the past years (45). All these virulence genes are controlled by PrfA, a central transcription regulator, which activates gene transcription by binding to a specific site termed the PrfA box and recruiting RNA polymerase to the PrfAdependent promot...