Lactococcus lactis is known to be capable of respiration under aerobic conditions in the presence of haemin. In the present study the effect of respiration on ATP production during growth on different sugars was examined. With glucose as the sole carbon source, respiratory conditions in L. lactis MG1363 resulted in only a minor increase, 21%, in biomass yield. Since ATP production through substrate-level phosphorylation was essentially identical with and without respiration, the increased biomass yield was a result of energy-saving under respiratory conditions estimated to be 0.4 mol of ATP/mol of glucose. With maltose as the energy source, the increase in biomass yield amounted to 51% compared with an aerobic culture that lacked haemin. This higher ATP yield was obtained by redirecting pyruvate metabolism from lactate to acetate production, and from savings through respiration. However, even after subtracting these contributions, approx. 0.3 mol of ATP/mol of glucose remained unaccounted for. A similar response to respiratory conditions (0.2 mol of ATP/mol of glucose) was observed in a mutant that had a decreased glucose uptake rate during growth on glucose caused by disruption of the PTSmannose (glucose/mannose-specific phosphotransferase system). Amino acid catabolism could be excluded as the source of the additional ATP. Since mutants without a functional H+-ATPase produced less ATP under sugar starvation and respiratory conditions, the additional ATP yield appears to come partly from energy saved on proton pumping through the H+-ATPase due to respiration and partly from a reversed function of the H+-ATPase towards oxidative phosphorylation. These results may contribute to the design and implementation of carbon-efficient high-cell-density cultures of this industrially important species of bacterium.