The function of type II NADH dehydrogenase (NDH-2) in Gram-positive Corynebacterium glutamicum was investigated by preparing strains with ndh, the NDH-2 gene, disrupted and over-expressed. Although disruption showed no growth defects on glucose minimum medium, the growth rate of the over-expressed strain was lower compared with its parent, C. glutamicum KY9714. Ndh-disruption and over-expression did not lead to a large change in the respiratory chain and energetics, including the cytochrome components and the H+/O ratio. However, in the strain that lacked NDH-2, membrane L-lactate oxidase activity increased, while NDH-2 over-expression led to decreased L-lactate and malate oxidase activities. In addition, relatively high cytoplasmic lactate dehydrogenase (LDH) activity was always present as was malate dehydrogenase, irrespective of NDH-2 level. Furthermore, L-lactate or malate-dependent NADH oxidase activity could be reproduced by reconstitution with the membranes and the cytoplasmic fraction isolated from the disruptant. These results suggest that coupling of LDH and the membrane L-lactate oxidase system, together with the malate-dependent NADH oxidase system, operates to oxidize NADH when the NDH-2 function is defective in C. glutamicum.