In a previous study it has been shown that the free moisture content in freeze-dried BCG vaccine dispensed in vials sealed with rubber stoppers increased during storage. The search for the source of this increase led us to explore the possibility that this additional moisture could originate from the rubber stoppers themselves. Therefore, the water permeability of various rubber stoppers has been studied, and the water content of grey butyl stoppers during some operations (autoclaving, oven-drying, freeze-drying, storage) used in the manufacturing of BCG vaccine has been determined. Our experiments showed: rapid water uptake during steam-autoclaving and rapid water release during subsequent oven-drying of the stoppers; a slow water uptake of the stoppers during freeze-drying and a slow water permeation through the stoppers when vials containing Indicating Drierite were stored in a water-saturated atmosphere. Among 12 types of rubber stoppers tested, the grey butyl stoppers and the silicone stoppers showed the lowest water uptake. Moisture-resistant wrappings decreased significantly the moisture uptake of Drierite. To delay moisture from reaching the vaccine it is recommended that the stoppers employed be as dry as possible.