We have obtained direct evidence indicating the presence of pyruvate-carboxylating activity in Corynebacterium glutamicum, a lysine-overproducing bacterium. This evidence was obtained through the use of C-13 nuclear magnetic resonance (NMR) spectroscopy and gas chromatography/mass spectrometry (GC-MS) of secreted metabolites in a lysine fermentation. The distribution of C-13 label after multiple turns in the tricarboxylic acid cycle was accounted for properly to obtain predictions for [C-13] metabolite enrichments that were employed in the interpretation of C-13-NMR and GC-MS data. Of critical importance in arriving at the conclusions was the use of C. glutamicum mutants with deletions of the pyruvate kinase and/or phosphoenolpyruvate carboxylase enzymes. Our results demonstrate the presence of pyruvate-carboxylating pathway(s) in C. glutamicum operating simultaneously with phosphoenolpyruvate carboxylase, with the latter enzyme contributing approximately 10% of the total oxaloacetate synthesis during the lysine-production phase with pyruvate and gluconate as carbon sources. These findings are important for developing strategies to increase the total carbon flux for synthesis of amino acids of the aspartate family through metabolic engineering.