The PhoP/PhoQ two-component system of Salmonella typhimurium governs transcription of some 25 loci in response to the extracellular concentration of Mg2+. We have now identified one of these loci as pmrCAB, which codes for a two-component system that mediates resistance to the antibiotic polymyxin B. Transcription of seven of 25 PhoP-activated loci was dependent on a functional PmrA protein, the response regulator of the PmrA/PmrB system. Expression of the PmrA-dependent loci was induced by either Mg2+ limitation or mild acidification, whereas transcription of a PmrA-independent gene was activated by Mg2+ limitation but not acid pH. Induction of PmrA-activated genes by Mg2 limitation required the PhoP and PhoQ proteins. In contrast, the acid-mediated activation of PmrA-regulated genes occurred in strains that were missing either one of these proteins. Transcriptional regulation by a cascade of two-component systems allows pathogenic bacteria to express their virulence determinants in response to a broader spectrum of environmental cues.