The influence of dimethyl sulfoxide (DMSO) on membrane thickness, multilamellar repeat distance, and phase transitions of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) was investigated by X-ray diffraction and small-angle neutron scattering (SANS). The differential scanning calorimetry (DSC) study of water freezing and ice melting was performed in the ternary DPPC/DMSO/water and binary DMSO/water systems. The methods applied demonstrated the differences in membrane structure in three sub-regions of the DMSO mole fraction (X DMSO ): from 0.0 to 0.3 for the first, from 0.3 to 0.8 for the second, and from 0.9 to 1.0 for the third sub-region. The thickness of the intermembrane solvent at T =20°C decreases from 14.4±1.8 Å at X DMSO =0.0 to 7.8±1.8 Å at X DMSO =0.1. The data were used to determine the number of free water molecules in the intermembrane space in the presence of DMSO. The results for 0.0≤X DMSO ≤0.3 were explained in the framework of DMSO-induced dehydration of the intermembrane space.