Aims: The effect of immobilization and long-term continuous culture was studied on probiotic and technological characteristics of lactic acid and probiotic bacteria. Methods and Results: A continuous culture in a two-stage system was carried out for 17 days at different temperatures ranging from 32 to 37degreesC, with a first reactor containing Bifidobacterium longum ATCC 15707 and Lactococcus lactis subsp. lactis biovar. diacetylactis MD immobilized separately in gel beads, and a second reactor operated with free cells released from the first reactor. The tolerance of free cells from both strains produced in the effluent medium of both reactors to hydrogen peroxide, simulated gastric and intestinal juices, antibiotics and nisin, and freeze-drying markedly increased with culture time and was generally higher after 6 days than that of stationary-phase cells produced during free-cell batch fermentations. The reversibility of the acquired tolerance of B. longum, but not L. diacetylactis, to antibiotics was shown during successive free-cell batch cultures. Conclusions: Free cells produced from continuous immobilized-cell culture exhibited altered physiology and increased tolerance to various chemical and physico-chemical stresses. Significance and Impact of the Study: Continuous culture with immobilized cells could be used to produce probiotic and lactic acid bacteria with enhanced technological and probiotic characteristics.