The PhoP-PhoQ two-component system is present in a number of Gram-negative bacteria where it has roles in Mg(2+) homeostasis and virulence. PhoQ is a transmembrane histidine kinase that activates PhoP-mediated regulation of a set of genes when the extracellular concentration of divalent cations is low. Divalent cations are thought to interact directly with the periplasmic PhoQ sensor domain. The PhoP-PhoQ systems of Escherichia coli and Pseudomonas aeruginosa are similar in their biological response to extracellular divalent cations; however, their sensor domains display little sequence identity. Here we have begun to explore the consequences of this sequence divergence by comparing the biophysical properties of the P. aeruginosa PhoQ sensor domain with the corresponding E. coli sensor domain. Unlike the E. coli protein, the P. aeruginosa PhoQ sensor domain undergoes changes in the circular dichroism and fluorescence spectra as well as destabilization of its dimeric form in response to divalent cations. These results suggest that distinct mechanisms of signal detection are utilized by these proteins. A hybrid protein in which the E. coli sensor domain has been substituted with the corresponding P. aeruginosa sensor domain responds normally to the presence of extracellular divalent cations in vivo in E. coli. Thus, despite apparent differences in the structural response to its stimulus, the P. aeruginosa sensor domain transduces signals to the E. coli PhoQ cytoplasmic kinase domain in a manner that mimics normal E. coli PhoQ function.