Abstract: Effect of banana, soybean, pearl barley powders and nonfat dry milk on the viability of freeze-dried Lactobacillus acidophilus at 4 and 25°C was studied during 30 days of storage. The survival of freeze-dried L. acidophilus at 4°C was greater than that at 25°C. The survival of L. acidophilus in banana, pearl barley, soybean powders and nonfat dry milk powder was higher than that in control (0.1% peptone water) at both temperatures indicating that addition of banana, soybean and pearl barley powders improved survival of L. acidophilus. The survival of L. acidophilus at 25°C in banana, pearl barley, soybean extract and nonfat dry milk was up to 6, 16, 20 and 25 days, respectively, while the organisms survived in samples stored at 4°C. The variation of viability may relate to hygroscopicity of different powders. Similar to nonfat dry milk, soybean powder preserved freeze-dried L. acidophilus during storage. The present study showed the potential of banana, soybean and pearl barley powders as cryoprotectants in freeze-dried probiotic preparations.