Analyses indicate that the lysine yield in Corynebacterium glutamicum is limited by suboptimal flux partitioning at either the glucose 6-phosphate (Glc6P), phosphoenolpyruvate (PEP), or pyruvate (Pyr) branch points (or a combination thereof), which results in disproportionate production of the required lysine precursors. Suboptimal flux partitioning at a metabolic branch point can result from the inadequate enzyme activity of a subordinate branch (a weakly rigid branch point) or from the active feedback regulation of the flux partitioning (strongly rigid branch point). In order to examine branch-point characteristics, we have utilized metabolite balances, constrained by biochemistry, to estimate flux distributions in the primary metabolic network of C. glutamicum from measured, extracellular, metabolite accumulation rates. These flux distributions, in combination with local metabolic perturbations, were used to infer branch-point characteristics. This study examines the flexibility of the Glc6P branch point, which could limit lysine yield via inadequate NADPH production, by perturbations induced from attenuation of Glc6P isomerase activity (first branch-point enzyme of glycolysis) and by fermentation on gluconate, which effectively bypasses the Glc6P branch point. Results from the analyses of these perturbations, as well as the flux distributions at the Glc6P branch point during the different phases of a control lysine fermentation, indicate that the Glc6P branch point is neither weakly nor strongly rigid, so that limitations in lysine yield must result from rigidity at either the PEP or Pyr branch point.