Aims: To investigate the effects of fermentation parameters on the cell growth and on the resistance to each step of the freeze-drying process of Lactococcus lactis subsp. lactis TOMSC161, a natural cheese isolate, using a response surface methodology. Methods and Results: Cells were cultivated at different temperatures (22, 30 and 38 degrees C) and pH (5.6, 6.2 and 6.8) and were harvested at different growth phases (0, 3 and 6 h of stationary phase). Cultivability and acidification activity losses of Lc. lactis were quantified after freezing, drying, 1 and 3 months of storage at 4 and 25 degrees C. Lactococcus lactis was not damaged by freezing but was sensitive to drying and to ambient temperature storage. Moreover, the fermentation temperature and the harvesting time influenced the drying resistance of Lc. lactis. Conclusions: Lactococcus lactis cells grown in a whey-based medium at 32 degrees C, pH 6.2 and harvested at late stationary phase exhibited both an optimal growth and the highest resistance to freeze-drying and storage. Significance and Impact of the Study: A better insight on the individual and interaction effects of fermentation parameters made it possible the freeze-drying and storage preservation of a sensitive strain of technological interest. Evidence on the particularly damaging effect of the drying step and the high-temperature storage is presented.