ABSTRACTTwo parameters of a one step freeze-drying process, cooling rate and protecting media, are studied in an effort to improve the viability and the stability of the preserved fungal strains. Cooling rates of 1.6 C, 3 C and 40 C/min are applied on cells ofSaccharomyces cerevisiaeandBrettanomyces bruxellensisand on spores ofTrichoderma virideandArthrobotrys arthrobotryoidespreserved in 93 suspending media containing polymers, sugars, albumin, milk, honey, polyols, amino acids alone or in combination. The viability rate ofSaccharomyces cerevisiaecells is increased from 30% to 96–98% by using an appropriate protecting medium containing 10% skim milk with 2 compounds among honey, sodium glutamate, trehalose or raffinose in the freeze-drying process carried out at a 3 C/min cooling rate. In the same conditionsArthrobotrys arthrobotryoidesspores, the most sensitive strain among the four tested, provides 60–65% viability, while this strain does not survive a classical freeze-drying in 10% skim milk. Moreover, with the improved method the stability of properties ofPenicillium expansumis demonstrated.