The response of Lactobacillus delbrueckii subsp. bulgaricus cells to heat stress was studied by use of a chemically defined medium. Two-dimensional electrophoresis (2-DE) analysis was used to correlate the kinetics of heat shock protein (HSP) induction with cell recovery from heat injury. We demonstrated that enhanced viability, observed after 10 min at 65°C, resulted from the overexpression of HSP and from mechanisms not linked to protein synthesis. In order to analyze the thermoadaptation mechanisms involved, thermoresistant variants were selected. These variants showed enhanced constitutive tolerance toward heat shock. However, contrary to the wild-type strain, these variants were poorly protected after osmotic or heat pretreatments. This result suggests that above a certain threshold, cells reach a maximum level of protection that cannot be easily exceeded. A comparison of protein patterns showed that the variants were able to induce more rapidly their adaptive mechanisms than the original strain. In particular, the variants were able to express constitutively more HSP, leading to the higher level of thermoprotection observed. This is the first report of the study by 2-DE of the heat stress response in L. delbrueckii subsp. bulgaricus.Lactobacillus delbrueckii subsp. bulgaricus is one of the most important industrial lactic acid bacteria. This fact is mainly due to its use in yogurt and fermented milk for human consumption. However, the price of current commercial preparations restricts its use, mainly because of the high susceptibility of L. delbrueckii subsp. bulgaricus to preservation processes. For example, this species would be a very interesting probiotic (40) if the production cost of these sensitive bacteria could be decreased substantially. This goal could be achieved by increasing cell survival or by using a less deleterious production process. Direct spray drying of vegetative microbial cells is emerging (3) as an efficient and inexpensive preservation method with a high output and may lower this cost. Knowledge of the resistance of these bacteria to heat stress would be helpful for mastering cell viability during this dehydration treatment. Teixeira et al. (35) studied L. delbrueckii subsp. bulgaricus thermotolerance and clearly showed the successful application of such a preservation method to concentrated suspensions of these bacteria (34,35). When cells were submitted to a heat pretreatment at 50°C or to a hyperosmotic pretreatment before lethal temperature challenge (65°C), the viability of the cells increased (13). Nevertheless, little is known of potential methods for enhancing cell survival and mechanisms used by the cell to protect itself from heat stress or to repair damage. Two-dimensional electrophoresis (2-DE) of proteins is currently one of the highest-resolution analytical techniques available for the study of protein expression patterns (the promising genome-wide DNA arrays are not yet available for L. delbrueckii subsp. bulgaricus). New technological developments have improved r...