Three isogenic strains of Lactococcus lactis with different levels of H2O-forming NADH oxidase activity were used to study the effect of oxygen on glucose metabolism: the parent strain L. lactis MG1363, a NOX- strain harboring a deletion of the gene coding for H2O-forming NADH oxidase, and a NOX strain with the NADH oxidase activity enhanced by about 100-fold. A comprehensive description of the metabolic events was obtained by using 13C nuclear magnetic resonance in vivo. The most noticeable results of this study are as follows: (i) under aerobic conditions the level of fructose 1,6-bisphosphate [Fru(1,6)P2] was lower than the level under anaerobic conditions, and the rate of Fru(1,6)P2 depletion was very high; (ii) the levels of 3-phosphoglycerate and phosphoenolpyruvate were considerably enhanced under aerobic conditions and significantly lower in the NOX- strain; and (iii) the glycolytic flux decreased in the presence of saturating levels of oxygen, but it was not altered in response to changes in the NADH oxidase activity. In particular, the observation that the glycolytic flux was not enhanced in the NOX strain indicated that glycolytic flux was not primarily determined by the level of NADH in the cell. The patterns of end products were identical for the NOX- and parent strains; in the NOX strain the carbon flux was diverted to the production of -acetolactate-derived compounds, and at a low pH this strain produced diacetyl at concentrations up to 1.6 mM. The data were integrated with the goal of identifying the main regulatory aspects of glucose metabolism in the presence of oxygen.