The metabolism of glucose by nongrowing cells of L. lactis strain MG5267 was studied under controlled conditions of pH, temperature, and gas atmosphere (anaerobic and aerobic) using a circulating system coupled to nuclear magnetic resonance (NMR) detection that allowed a noninvasive determination of intracellular pools of intermediate metabolites by C-13- NMR with a time resolution of 30 seconds. In addition, intracellular parameters, such as pH, NTP levels, and concentration of inorganic phosphate in the cytoplasm, could be monitored on-line by P-31-NMR with a time resolution of approx. 3 min. The time course for the concentrations of intracellular fructose 1,6-bisphosphate (FBP), S-phosphoglycerate (3-PGA), and phosphoenolpyruvate (PEP), together with kinetic measurements of substrate consumption and endproducts formation, were used as a basis for the construction of a mechanistic model for glycolysis. In vivo measurements were complemented with determinations of phosphorylated metabolites in perchloric acid extracts. A top-down model was developed by simplifying the metabolism to the resolution allowed by the experimental data collected by in vivo NMR (grouped in seven metabolic steps). This simplified mechanistic model was adjusted to the metabolite concentrations determined by in vivo NMR. The results obtained led to the rationalization of the dynamics of glucose metabolism as being driven largely by ATP surplus. This excess causes accumulation of FBP due to NAD(+) limitation, whose regeneration is dependent on downstream pyruvate reduction. The model was capable of predicting qualitative shifts in the metabolism of glucose when changing from anaerobic to aerobic conditions.