The involvement of nicotinamide adenine nucleotides (NAD+, NADH) in the regulation of glycolysis in Lactococcus lactis was investigated by using13C and 31P NMR to monitor in vivothe kinetics of the pools of NAD+, NADH, ATP, inorganic phosphate (Pi), glycolytic intermediates, and end products derived from a pulse of glucose. Nicotinic acid specifically labeled on carbon 5 was synthesized and used in the growth medium as a precursor of pyridine nucleotides to allow for in vivo detection of13C-labeled NAD+ and NADH. The capacity ofL. lactis MG1363 to regenerate NAD+ was manipulated either by turning on NADH oxidase activity or by knocking out the gene encoding lactate dehydrogenase (LDH). An LDH−deficient strain was constructed by double crossover. Upon supply of glucose, NAD+ was constant and maximal (∼5 mm) in the parent strain (MG1363) but decreased abruptly in the LDH− strain both under aerobic and anaerobic conditions. NADH in MG1363 was always below the detection limit as long as glucose was available. The rate of glucose consumption under anaerobic conditions was 7-fold lower in the LDH− strain and NADH reached high levels (2.5 mm), reflecting severe limitation in regenerating NAD+. However, under aerobic conditions the glycolytic flux was nearly as high as in MG1363 despite the accumulation of NADH up to 1.5 mm. Glyceraldehyde-3-phosphate dehydrogenase was able to support a high flux even in the presence of NADH concentrations much higher than those of the parent strain. We interpret the data as showing that the glycolytic flux in wild type L. lactis is not primarily controlled at the level of glyceraldehyde-3-phosphate dehydrogenase by NADH. The ATP/ADP/Pi content could play an important role.