The integration host factor (IHF) is a DNA-binding and -bending protein with roles in local DNA structural organization and transcriptional regulation in Gram-negative bacteria. This heterodimeric protein is composed of the two highly homologous subunits IHFα and IHFβ. DNA microarray analysis was used to define the regulon of genes subject to IHF control in Salmonella enterica serovar Typhimurium (S. Typhimurium). The transcription profile of the wild type was compared with those of mutants deficient in IHFα, IHFβ, or both IHFα and IHFβ. Our data reveal a new connection between IHF and the expression of genes required by the bacterium to undergo the physiological changes associated with the transition from exponential growth to stationary phase. When a mutant lacking IHF entered stationary phase, it displayed downregulated expression of classic stationary-phase genes in the absence of any concomitant change in expression of the RpoS sigma factor. Purified IHF was found to bind to the regulatory regions of stationary-phase genes indicating an auxiliary and direct role for IHF in RpoS-dependent gene activation. Loss of IHF also had a profound influence on expression of the major virulence genes and epithelial cell invasion, indicating a role in co-ordinating regulation of the pathogenic traits with adaptation to stationary phase. Although the three mutants showed considerable overlaps in the genes affected by the ihf lesions, the observed patterns were not identical, showing that S. Typhimurium has not one but three overlapping IHF regulons.