The aim of this study was to investigate the effects of protective media and different relative vapour pressures (RVPs) on the survival of probiotics during freeze drying and subsequent storage, to determine the optimal conditions for the production of freeze dried probiotics at industrial scale, ensuring a high survival rate. The effect of protective media i.e. reconstituted skimmed milk (RSM) or either of the cryoprotective disaccharides lactose, trehalose, sucrose, maltose, lactose + maltose and lactose + trehalose on the survival of a probiotic culture was assessed at five different RVP environments (0.0%, 11.4%, 33.2%, 44.1% and 76.1%) at room temperature in freeze-dried systems. RVP was shown to have a significant effect on the survival rates of the probiotic cultures Lactobacillus paracasei NFBC 338 and Lactobacillus rhamnosus GG following freeze drying in RSM. Interestingly, retention of cell viability was greatest for cells stored at 11.4% RVP, but was compromised at all other RVPs tested. However, an increased tolerance to freeze drying was observed for L. rhamnosus GG when dried in the presence of disaccharides in the order of trehalose = lactose + maltose >= lactose + trehalose >= maltose > lactose > sucrose. Survival studies over a 38-40 day storage period indicated that trehalose and lactose + maltose were the most effective cryoprotective additives, especially notable at 0.0 and 11.4% RVP. At all other RVPs tested, viability was compromised. Crystallisation of the disaccharides was observed to be a detrimental factor affecting the survival of Lactobacillus during storage at high RVP, where an inverse relationship was shown to exist between the % RVP and the glass transition temperature (T-g) of the disaccharides.