Previous studies have demonstrated that the water content of freeze-dried vaccines increases during storage. The reasons for these variations in water content are discussed in this paper. Different possible mechanisms have been considered: microleakages at the closure sealing point, water vapour transfer through the closure (permeation, loss and uptake of water by the stopper). Several types of vials and closures were studied, under different conditions of storage. Regardless of the vial form selected, the probability of microleakages was low. The stopper would seem to play an important role in the water transfer mechanisms. The moisture content of the stopper determines its equilibrium relative humidity (ERH) and whether there will be moisture transfer between the stopper and the vial contents and between the stopper and the storage atmosphere.