The differences in the packing of the bilayer interface in lipid vesicles of different sizes have been measured by determining the dimerization equilibrium (K-dapp) Of merocyanine 540 (MC540) incorporated to lipid bilayers. The size was changed by preparing lipid vesicles in isosmotic conditions by extrusion through polycarbonate membranes of pore size of 100, 200, and 400 nm and by introducing an osmotic shrinkage with polethylene glycol (PEG). In the absence of osmosis, the packing of dipalmitoylphosphatidylcholine (DPPC) bilayers decreases with the decrease in the vesicle diameter. In contrast, when the vesicle size is decreased by inducing a shrinkage, an increase in the packing of the bilayer interface was observed. A correlation was found between the packing increase with the osmotic shock with SUV's and 100, 200, and 400 nm LUV's : the larger the vesicle, the larger was the effect of osmosis on the packing. Experiments with vesicles of 100 nn with the PEG evenly distributed inside and outside showed a 3-fold higher K-dapp. In contrast, the effect on K-dapp of ethylene glycol, a molecule that permeates the bilayer, was shown to be negligible. This suggests that the volume decrease induced by the osmotic shrinkage leads to changes in the lateral packing of the membrane interface by effecting the cohesion forces. This effect is comparable to that found when, in the absence of osmosis, the packing increases with the length of the hydrocarbon chain for a given diameter. The fact that this conclusion can be derived using a dye that dimerizes at the membrane interface allows to conclude that the osmotic stress affects the packing by extrusion of the water layers adjacent to polar groups. In contrast, spontaneous curvature can show defects in the surface packing.