Entry into intestinal epithelial cells is an essential feature in the pathogenicity of Salmonella typhi, which causes typhoid fever in humans. This process requires intact motility and secretion of the invasion-promoting Sip proteins, which are targets of the type III secretion machinery encoded by the inv, spa and prg loci. During our investigations into the entry of S. typhi into cultured epithelial cells, we observed that the secretion of Sip proteins and flagellin was impaired in Vi-expressing strains. We report here that the production of Sip proteins, flagellin and Vi antigen is differentially modulated by the RcsB-RcsC regulatory system and osmolarity. This regulation occurs at both transcriptional and post-translational levels. Under low-osmolarity conditions, the transcription of iagA, invF and sipB genes is negatively controlled by the RcsB regulator, which probably acts in association with the viaB locus-encoded TviA protein. The cell surface-associated Vi polysaccharide, which was maximally produced under these growth conditions, prevented the secretion of Sip proteins and flagellin. As the NaCl concentration in the growth medium was increased, transcription of iagA, invF and sipB was found to be markedly increased, whereas transcription of genes involved in Vi antigen biosynthesis was greatly reduced. The expression of iagA, whose product is involved in invF and sipB transcription, occurred selectively during the exponential growth phase and was maximal in the presence of 300mM NaCl. At this osmolarity, large amounts of Sips and flagellin were secreted in culture supernatants. As expected from these results, and given the essential role of Sip proteins and motility in entry, RcsB and osmolarity modulated the invasive capacity of S. typhi. Together, these findings might reflect the adaptive response of S. typhi to the environments encountered during the different stages of pathogenesis.