During infection of mammalian hosts, facultative intracellular pathogens have to adjust rapidly to different environmental conditions encountered during passage through the gastrointestinal tract and following uptake into epithelial cells and macrophages. Successful establishment within the host therefore requires the co-ordinated expression of a large number of virulence genes necessary for the adaptation between the extracellular and intracellular phases of infection. In this study we show that the bacterial signal molecule, ppGpp, plays a major role in mediating the environmental signals involved in the regulation of both the extracellular (iuvasion) and intracellular virulence gene programs. Under oxygen-limiting conditions, we observed a strong ppGpp-dependence for invasion gene expression, the result of severe reductions in expression of the Salmonella pathogenicity island 1 (SPI1) transcriptional regulator genes hilA, C, D and invF. Over-expression of the non-SPI1-encoded regulator, RtsA, restored hilA expression in the absence of ppGpp. SPI2-encoded genes, required for intracellular proliferation in macrophages, were activated in the wildtype strain under aerobic, late-log phase growth conditions. The expression of SPI2 genes was also shown to be ppGpp-dependent under these conditions. The results from this study suggest a mechanism for the alternate regulation of the opposing extracellular and intracellular virulence gene programs, and indicate a remarkable specificity for ppGpp in the regulation of genes involved in virulence compared to the rest of the genome. This is the first demonstration that this highly conserved regulatory system is involved in bacterial virulence gene expression on a global scale.