Objective: Dynamic changes in intracellular pH regulate diverse cell processes, including proliferation, migration, and transformation. However, our understanding of how physiological changes in pH affect protein activities and macromolecular assemblies driving specific cell behaviors is limited. We are studying the structure and function of "pH sensors", or proteins with activities or ligand-binding affinities that are regulated by physiological changes in pH. Methods: Our collaborative studies include constant pH molecular dynamics simulations, NMR spectroscopy, biochemical analyses, and cell function to characterize pH sensors regulating directed cell migration, cell cycle progression, glycolysis, and tumorigenesis. Results: We identified three pH sensors regulating cell migration; we found that pH-dependent histidine switches determine phosphoinositide binding by Dbs, a guanine nucleotide exchange factor activating Cdc42 for cell polarity, and by cofilin to promote actin filament assembly for membrane protrusion, and also that pH-dependent allosteric regulation of talin binding to actin filaments determines remodelling of focal adhesions for migratory rate. For cell cycle progression, our data suggest that Wee1 kinase may be a pH sensor regulating pH-dependent timing of G2/M. We found that the protonation state of His350 in the kinase domain of human Wee1 determines orientation of the C-helix, with His350+ inducing an active conformation and His350 neutral inducing an inactive conformation. For glycolysis, we are studying phosphofructokinase-1, which directly binds to the Na-H exchanger NHE1 and has a 200-fold increase in activity from pH 7.0 to 7.4. For tumorigenesis, we are using the Drosophila rough eye phenotype to identify pH sensors necessary for oncogene-induced dysplasia. These studies are defining the structural basis and functional significance of proteins that are regulated by physiological changes in pH to drive specific cell behaviors.