As enteric pathogens, the salmonellae have developed systems by which they can sense and adapt appropriately to deleterious intestinal components that include bile. Previously, growth in the presence of bile was shown to repress the transcription of prgH, a locus encoding components of the Salmonella pathogenicity island I (SPI-1) type III secretion system (TTSS) necessary for eukaryotic cell invasion. This result suggested an existing interaction between salmonellae, bile, and eukaryotic cell invasion. Transcription assays demonstrated that invasion gene regulators (e.g.,sirC and invF) are repressed by bile. However, bile does not interact with any of the invasion regulators directly but exerts its effect at or upstream of the two-component system at the apex of the invasion cascade, SirA-BarA. As suggested by the repression of invasion gene transcription in the presence of bile, Western blot analysis demonstrated that proteins secreted by the SPI-1 TTSS were markedly reduced in the presence of bile. Furthermore, Salmonella enterica serovar Typhimurium grown in the presence of bile was able to invade epithelial cells at only 4% of the level of serovar Typhimurium grown without bile. From these data, we propose a model whereby serovar Typhimurium uses bile as an environmental signal to repress its invasive capacity in the lumen of the intestine, but upon mucous layer penetration and association with intestinal epithelial cells, where the apparent bile concentration would be reduced, the system would become derepressed and invasion would be initiated.