The PhoP/PhoQ two-component regulatory system governs the adaptation to low Mg(2+) environments and virulence in several Gram-negative species. During growth in low Mg(2+), the sensor PhoQ modifies the activity of the response regulator PhoP promoting gene transcription, whereas growth in high Mg(2+) represses transcription of PhoP-activated genes. The PhoQ protein harbors a periplasmic domain of 146 amino acid residues that binds Mg(2+) in vitro and is required for Mg(2+)-mediated repression in vivo. Here, we identify periplasmic mutants of the Salmonella PhoQ protein that allow transcription of PhoP-activated genes even under high Mg(2+) concentrations. When expressed in a strain harboring a PhoP variant that is phosphorylated from acetyl phosphate, some of the mutants failed to repress PhoP-promoted transcription in high Mg(2+), whereas others displayed a wild-type ability to do so. Mutant PhoQ proteins that allowed expression of PhoP-activated genes in high Mg(2+) displayed a pattern of iron-mediated cleavage in vitro that was different from that displayed by wild-type PhoQ, indicative of altered Mg(2+) binding. A PhoQ protein with the conserved histidine residue (H277) substituted by alanine could not promote transcription of PhoP-activated genes in low Mg(2+) but could turn off expression in response to high Mg(2+). Our studies demonstrate that residues G93, W97, H120 and T156 are required for a wild-type response to Mg(2+), and suggest that Mg(2+) binding to the periplasmic domain regulates several activities in the PhoQ protein.