Enable this bacterium to persist is imperative in the quest to understand E. faecalis pathogenicity. Previous reports suggest that enterococci control turgor by actively modulating the pool of osmotically active solutes in their cytoplasm, thereby allowing water content to be adjusted by osmosis. As part of a continued effort to decipher the various physiological aspects contributing to the success of this versatile pathogen, we here describe the global transcriptional profile of E. faecalis V583 upon the encounter with high concentrations of NaCl. The concentrations of the RNA samples were measured by using the NanoDrop, and the quality was assessed by using the RNA 600 Nano LabChip kit and the Bioanalyzer 2100. cDNA was synthesized and labeled with the Fairplay II Microarray labeling kit, with modifications as previously described. Labeled samples were then dried, prior to resuspension in 140 ml hybridization solution and hybridized as described. The microarray used in this work has also been described previously. Three replicate hybridizations were performed with three separate batches of RNA. The three batches of RNA were obtained in three separate growth experiments. The Cy3 and Cy5 dyes used during cDNA synthesis were swapped in one of the three replicate hybridizations. All samples were cohybridized with control samples collected at equal time points. The gene cluster consists of distinct modules predicted to be responsible for the sequential steps of the polysaccharide biosynthesis process, i.e. synthesis of dTDP-rhamnose, glycosyltransferase activity, polymerization and peptidoglycan-linkage, although the exact biochemical functions of the different genes have not been experimentally determined. The Epa polysaccharide has been investigated for its implication in virulence in various animal infection models, and has thus been considered as a vital virulence trait of E. faecalis. The induction of parts of the epa gene cluster during treatment with NaCl suggested that Epa may be involved in the osmotic stress response in E. faecalis. To further investigate this notion, a series of experiments providing unequivocal functional genetic evidence for the involvement of the epa locus in E. faecalis osmoprotection were designed using two different epa disruption mutants. The bacterial cell envelope provides essential protection from the external environment and confers strength and rigidity to counteract the effects of osmotic stress conditions on the cell. Furthermore, osmosensor activity is likely to be mediated through changes in membrane properties. However, if accumulated de novo synthesized rhamnose functioned as an osmoprotector, the salt resistance of the DepaB mutant strain would be expected to resemble that of the parent strain. Our data thus indicate that the entire Epa biosynthesis pathway, and not only the genes responsible for rhamnose biosynthesis.