Differential scanning calorimetry (DSC) has been used to study the effects of repeated freezing and thawing on dipalmitoylphosphatidylcholine (DPPC) vesicles. Aqueous suspensions of both multilamellar vesicles (MLVs) and large unilamellar vesicles (LUVs) were cycled between -37 and 8°C, and for each thawing event, the enthalpy of ice-melting was measured. In the case of MLVs, the enthalpy increased each time the vesicles were thawed until a steady state was attained. In contrast, the enthalpies measured for LUV suspensions were independent of the number of previous thawing events. It was concluded that MLVs in terms of freezing characteristics contain two pools of water, namely bulk water and interlamellar water. Interlamellar water does not freeze under the conditions employed in the present study, and the MLVs therefore experience freeze-induced dehydration, which is the reason for the observed increase in ice-melting enthalpy. Furthermore, the thermodynamic results suggest that the osmotic stress resulting from the freeze-induced dehydration changes the lamellarity of the MLVs.