In its natural environment, the soil, the Gram-positive model bacterium Bacillus subtilis frequently encounters nutrient limitation and other stress factors. Efficient adaptation mechanisms are necessary to cope with this wide range of environmental challenges. The ability to utilize diverse carbon sources represents a key adaptation process that allows B. subtilis to thrive in its natural habitat. To gain a comprehensive insight into the metabolism of B. subtilis, global metabolite analyses were performed during growth with glucose alone or glucose with either malate, fumarate or citrate as carbon/energy sources. Furthermore, to achieve a comprehensive coverage of a wide range of chemically different metabolites, complementary GC-MS, LC-MS and (1)H-NMR analyses were applied. This study reveals that the availability of different carbon sources results in different extracellular metabolite profiles whereas a regulated intracellular metabolite equilibrium was observed. In addition, the typical energy-starvation induced activation of the general stress sigma factor σ(B) was only observed upon entry into the stationary phase with glucose or glucose and malate as carbon sources.