Solutions of sucrose, trehalose, skim milk, sucrose+skim milk, trehalose+skim milk, sucrose+trehalose or sucrose+trehalose+skim milk were used as protective media for Lactobacillus salivarius subsp. Salivarius (UCC 500) in freeze-drying and the ability of these materials to stabilize the bacteria during subsequent storage was determined. A decrease of about 99% or more in the viable population was observed when no protective solute (distilled water only) was used. Trehalose greatly enhanced survival rate when used alone or together with other protective materials. Trehalose+sucrose in addition to skim milk were the most efficient materials giving a survival rate of 83–85% immediately after freeze-drying and enhanced stability during subsequent storage. The ability of Lactobacillus salivarius subsp. Salivarius for utilization of trehalose as a sole source of carbon was examined. This strain could ferment trehalose and generated a significant drop in pH from initial 6.8 to 4.8. The results however suggested that the capacity of Lactobacillus salivarius subsp. Salivarius to metabolize trehalose did not contribute to resistance to freeze-drying. The effect of water on survival rate was also examined. The results showed that a residual water content of 2.8 to about 5.6% improved survival rate during storage.