The intracellular metabolism of Listeria monocytogenes was studied by (13)C-isotopologue profiling using murine J774A.1 macrophages as host cells. Six hours after infection, bacteria were separated from the macrophages and hydrolyzed. Amino acids were converted into tert-butyl-dimethylsilyl derivatives and subjected to gas chromatography/mass spectrometry. When the macrophages were supplied with [U-(13)C(6)]glucose prior to infection, but not during infection, label was detected only in Ala, Asp and Glu of the macrophage and bacterial protein with equal isotope distribution. When [U-(13)C(6)]glucose was provided during the infection period, (13)C label was found again in Ala, Asp and Glu from host and bacterial protein, but also in Ser, Gly, Thr and Val from the bacterial fraction. Mutants of L. monocytogenes defective in the uptake and catabolism of the C(3)-metabolites, glycerol and/or dihydroxyacetone, showed reduced incorporation of [U-(13)C(6)]glucose into bacterial amino acids under the same experimental settings. The (13)C pattern suggests that (i) significant fractions (50-100%) of bacterial amino acids were provided by the host cell, (ii) a C(3)-metabolite can serve as carbon source for L. monocytogenes under intracellular conditions and (iii) bacterial biosynthesis of Asp, Thr and Glu proceeds via oxaloacetate by carboxylation of pyruvate.