The packing and polarity of dipalmitoylphosphatidylcholine unilamellar vesicles in the gel state were measured in the presence of trehalose and phloretin by using fluorescent probes inserted at different levels of the bilayer interface. The molecular probes used were Laurdan (LA), Prodan, (PA), Dansyl derivatives (DA), and Merocyanine 540 (MC540). The binding of trehalose to the carbonyl groups and of phloretin to the phosphate groups affects the packing and the polarity of the glycerol backbone region. This effect is more pronounced when the bilayers are dehydrated and rehydrated in the presence of the sugar, thus indicating that the effect is due to water replacement. The results can be interpreted in terms of the rearrangement of the water structure in the environment around the C=O and P=O groups.