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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 (P_i), 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 …