The Salmonella typhimurium transcriptional regulators, PhoP/PhoQ, induce phoP-activated gene (pag) expression to promote virulence and intracellular survival within macrophages. This response to the macrophage intracellular environment is simulated by phoP/phoQ constitutive mutations (phenotype PhoPc) that increase the expression of pag genes and repress the synthesis of approximately 20 proteins encoded by phoP-repressed genes (prg genes) (S. I. Miller and J. J. Mekalanos, J. Bacteriol. 172:2485-2490, 1990). PhoPc bacteria are attenuated for mouse virulence, suggesting that prg genes are virulence genes. We now report the identification of five unlinked prg loci by use of the transposon TnphoA. In general, medium conditions (i.e., starvation) that activate pag expression repress prg expression. However, variable effects on the PhoP regulon were observed when bacteria were grown under different oxygen tensions (pag and prg genes) or exposed to low pH (prg genes), suggesting heterogenous control of the regulon. One prg locus, prgH, was demonstrated to contribute to mouse virulence by both the oral and the intraperitoneal routes. prgH was located at 59 min on the Salmonella chromosome, a region where other genes essential to invasion of epithelial cells are clustered. The prgH locus was highly linked to one invasion locus, hil (C.A. Lee, B.D. Jones, and S. Falkow, Proc. Natl. Acad. Sci. USA 89:1847-1851, 1992), although transcription of prgH was opposite that of the Tn5B50-encoded promoters that result in a hyperinvasive or hil phenotype. Both PrgH and PhoPc mutant S. typhimurium were found to be defective in induction of endocytosis by Madin-Darby canine kidney (MDCK) epithelial cells. The invasion defect of PrgH but not that of PhoPc mutant bacteria was complemented by plasmids containing prgH (hil) DNA. Therefore, two virulence properties of Salmonella species, induction of endocytosis by epithelial cells and survival within macrophages, are oppositely modulated by the PhoP/PhoQ virulence regulators.