Salmonella typhimurium causes enteric and systemic disease by invading the intestinal epithelium of the distal ileum, a process requiring the invasion genes of Salmonella pathogenicity island 1 (SPI-1). BarA, a sensor kinase postulated to interact with the response regulator SirA, is required for the expression of SPI-1 invasion genes. We found, however, that a barA null mutation had little effect on virulence using the mouse model for septicaemia. This confounding result led us to seek environmental signals present in the distal ileum that might supplant the need for BarA. We found that acetate restored the expression of invasion genes in the barA mutant, but had no effect on a sirA mutant. Acetate had its effect only at a pH that allowed its accumulation within the bacterial cytoplasm and not with the deletion of ackA and pta, the two genes required to produce acetyl-phosphate. These results suggest that the rising concentration of acetate in the distal ileum provides a signal for invasion gene expression by the production of acetyl-phosphate in the bacterial cytoplasm, a pathway that bypasses barA. We also found that a Delta(ackA-pta) mutation alone had no effect on virulence but, in combination with Delta(barA), it increased the oral LD50 24-fold. Thus, the combined loss of the BarA- and acetate-dependent pathways is required to reduce virulence. Two other short-chain fatty acids (SCFA), propionate and butyrate, present in high concentrations in the caecum and colon, had effects opposite to those of acetate: neither restored invasion gene expression in the barA mutant, and both, in fact, reduced expression in the wild-type strain. Further, a combination of SCFAs found in the distal ileum restored invasion gene expression in the barA mutant, whereas colonic conditions failed to do so and also reduced expression in the wild-type strain. These results suggest that the concentration and composition of SCFAs in the distal ileum provide a signal for productive infection by Salmonella, whereas those of the large intestine inhibit invasion.