When cells are exposed to changes in the osmotic pressure of the external medium, they respond with mechanisms of osmoregulation. An increase of the extracellular osmolality leads to the accumulation of internal solutes by biosynthesis or uptake. Particular bacterial transporters act as osmosensors and respond to increased osmotic pressure by catalyzing uptake of compatible solutes. The functions of osmosensing, osmoregulation , and solute transport of these transporters can be analyzed in molecular detail after solubilization, isolation, and reconstitution into phospholipid vesicles. Using this approach, intrinsic functions of osmosensing transporters are studied in a defined hydrophilic (access to both sides of the membrane) and hydrophobic surrounding (phospholipid membrane), and free of putative interacting cofactors and regulatory proteins.