The aim of the study was to study the role of carbon dioxide metabolism in Streptococcus thermophilus through investigation of the phenotype of a carbamoylphosphate synthetase-negative mutant. The effect of carbon dioxide on the nutritional requirements of Strep. thermophilus DSM20617(T) and its derivative, carbamoylphosphate synthetase-negative mutant A17(Delta carB), was investigated by cultivating the strain in a chemically defined medium under diverse gas compositions and in milk. The results obtained revealed that CO2 depletion or carB gene inactivation determined the auxotrophy of Strep. thermophilus for l-arginine and uracil. In addition, the parent strain grew faster than the mutant, even when milk was supplemented with uracil or arginine. Milk growth experiments underlined that carbamoylphosphate synthetase activity was essential for the optimal growth of Strep. thermophilus in milk. The study of the carbon dioxide metabolism in Strep. thermophilus revealed new insights with regard to the metabolism of this species, which could be useful for the optimization of dairy fermentation processes.