Streptococcus thermophilus is a major component of dairy starter cultures used for the manufacture of yoghurt and cheese. In this study, the CO(2) metabolism of S. thermophilus DSM 20617(T), grown in either a N(2) atmosphere or an enriched CO(2) atmosphere, was analysed using both genetic and proteomic approaches. Growth experiments performed in a chemically defined medium revealed that CO(2) depletion resulted in bacterial arginine, aspartate and uracil auxotrophy. Moreover, CO(2) depletion governed a significant change in cell morphology, and a high reduction in biomass production. A comparative proteomic analysis revealed that cells of S. thermophilus showed a different degree of energy status depending on the CO(2) availability. In agreement with proteomic data, cells grown under N(2) showed a significantly higher milk acidification rate compared with those grown in an enriched CO(2) atmosphere. Experiments carried out on S. thermophilus wild-type and its derivative mutant, which was inactivated in the phosphoenolpyruvate carboxylase and carbamoyl-phosphate synthase activities responsible for fixing CO(2) to organic molecules, suggested that the anaplerotic reactions governed by these enzymes have a central role in bacterial metabolism. Our results reveal the capnophilic nature of this micro-organism, underlining the essential role of CO(2) in S. thermophilus physiology, and suggesting potential applications in dairy fermentation processes.